WorldWideScience

Sample records for bacterial cell biotherapeutics

  1. Computationally driven deletion of broadly distributed T cell epitopes in a biotherapeutic candidate.

    Science.gov (United States)

    Salvat, Regina S; Parker, Andrew S; Guilliams, Andrew; Choi, Yoonjoo; Bailey-Kellogg, Chris; Griswold, Karl E

    2014-12-01

    Biotherapeutics are subject to immune surveillance within the body, and anti-biotherapeutic immune responses can compromise drug efficacy and patient safety. Initial development of targeted antidrug immune memory is coordinated by T cell recognition of immunogenic subsequences, termed "T cell epitopes." Biotherapeutics may therefore be deimmunized by mutating key residues within cognate epitopes, but there exist complex trade-offs between immunogenicity, mutational load, and protein structure-function. Here, a protein deimmunization algorithm has been applied to P99 beta-lactamase, a component of antibody-directed enzyme prodrug therapies. The algorithm, integer programming for immunogenic proteins, seamlessly integrates computational prediction of T cell epitopes with both 1- and 2-body sequence potentials that assess protein tolerance to epitope-deleting mutations. Compared to previously deimmunized P99 variants, which bore only one or two mutations, the enzymes designed here contain 4-5 widely distributed substitutions. As a result, they exhibit broad reductions in major histocompatibility complex recognition. Despite their high mutational loads and markedly reduced immunoreactivity, all eight engineered variants possessed wild-type or better catalytic activity. Thus, the protein design algorithm is able to disrupt broadly distributed epitopes while maintaining protein function. As a result, this computational tool may prove useful in expanding the repertoire of next-generation biotherapeutics.

  2. Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry

    OpenAIRE

    Doneanu, Catalin E; Xenopoulos, Alex; Fadgen, Keith; Murphy, Jim; Skilton, St. John; Prentice, Holly; Stapels, Martha; Chen, Weibin

    2012-01-01

    Assays for identification and quantification of host-cell proteins (HCPs) in biotherapeutic proteins over 5 orders of magnitude in concentration are presented. The HCP assays consist of two types: HCP identification using comprehensive online two-dimensional liquid chromatography coupled with high resolution mass spectrometry (2D-LC/MS), followed by high-throughput HCP quantification by liquid chromatography, multiple reaction monitoring (LC-MRM). The former is described as a “discovery” assa...

  3. Profiling the effects of process changes on residual host cell proteins in biotherapeutics by mass spectrometry.

    Science.gov (United States)

    Schenauer, Matthew R; Flynn, Gregory C; Goetze, Andrew M

    2013-01-01

    An advanced liquid chromatography/mass spectrometry (MS) platform was used to identify and quantify residual Escherichia coli host cell proteins (HCPs) in the drug substance (DS) of several peptibodies (Pbs). Significantly different HCP impurity profiles were observed among different biotherapeutic Pbs as well as one Pb purified via multiple processes. The results can be rationally interpreted in terms of differences among the purification processes, and demonstrate the power of this technique to sensitively monitor both the quantity and composition of residual HCPs in DS, where these may represent a safety risk to patients. The breadth of information obtained using MS is compared to traditional multiproduct enzyme-linked immunosorbent assay (ELISA) values for total HCP in the same samples and shows that, in this case, the ELISA failed to detect multiple HCPs. The HCP composition of two upstream samples was also analyzed and used to demonstrate that HCPs that carry through purification processes to be detectable in DS are not always among those that are the most abundant upstream. Compared to ELISA, we demonstrate that MS can provide a more comprehensive, and accurate, characterization of DS HCPs, thereby facilitating process development as well as more rationally assessing potential safety risks posed by individual, identified HCPs. © 2013 American Institute of Chemical Engineers.

  4. Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry.

    Science.gov (United States)

    Doneanu, Catalin E; Xenopoulos, Alex; Fadgen, Keith; Murphy, Jim; Skilton, St John; Prentice, Holly; Stapels, Martha; Chen, Weibin

    2012-01-01

    Assays for identification and quantification of host-cell proteins (HCPs) in biotherapeutic proteins over 5 orders of magnitude in concentration are presented. The HCP assays consist of two types: HCP identification using comprehensive online two-dimensional liquid chromatography coupled with high resolution mass spectrometry (2D-LC/MS), followed by high-throughput HCP quantification by liquid chromatography, multiple reaction monitoring (LC-MRM). The former is described as a "discovery" assay, the latter as a "monitoring" assay. Purified biotherapeutic proteins (e.g., monoclonal antibodies) were digested with trypsin after reduction and alkylation, and the digests were fractionated using reversed-phase (RP) chromatography at high pH (pH 10) by a step gradient in the first dimension, followed by a high-resolution separation at low pH (pH 2.5) in the second dimension. As peptides eluted from the second dimension, a quadrupole time-of-flight mass spectrometer was used to detect the peptides and their fragments simultaneously by alternating the collision cell energy between a low and an elevated energy (MSE methodology). The MSE data was used to identify and quantify the proteins in the mixture using a proven label-free quantification technique ("Hi3" method). The same data set was mined to subsequently develop target peptides and transitions for monitoring the concentration of selected HCPs on a triple quadrupole mass spectrometer in a high-throughput manner (20 min LC-MRM analysis). This analytical methodology was applied to the identification and quantification of low-abundance HCPs in six samples of PTG1, a recombinant chimeric anti-phosphotyrosine monoclonal antibody (mAb). Thirty three HCPs were identified in total from the PTG1 samples among which 21 HCP isoforms were selected for MRM monitoring. The absolute quantification of three selected HCPs was undertaken on two different LC-MRM platforms after spiking isotopically labeled peptides in the samples. Finally

  5. Gut microbiota: next frontier in understanding human health and development of biotherapeutics

    Directory of Open Access Journals (Sweden)

    Prakash S

    2011-07-01

    Full Text Available Satya Prakash, Laetitia Rodes, Michael Coussa-Charley, Catherine Tomaro-DuchesneauBiomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering and Artificial Cells and Organs Research Centre, Faculty of Medicine, McGill University, Montreal, Quebec, CanadaAbstract: The gut microbiota is a remarkable asset for human health. As a key element in the development and prevention of specific diseases, its study has yielded a new field of promising biotherapeutics. This review provides comprehensive and updated knowledge of the human gut microbiota, its implications in health and disease, and the potentials and limitations of its modification by currently available biotherapeutics to treat, prevent and/or restore human health, and future directions. Homeostasis of the gut microbiota maintains various functions which are vital to the maintenance of human health. Disruption of the intestinal ecosystem equilibrium (gut dysbiosis is associated with a plethora of human diseases, including autoimmune and allergic diseases, colorectal cancer, metabolic diseases, and bacterial infections. Relevant underlying mechanisms by which specific intestinal bacteria populations might trigger the development of disease in susceptible hosts are being explored across the globe. Beneficial modulation of the gut microbiota using biotherapeutics, such as prebiotics, probiotics, and antibiotics, may favor health-promoting populations of bacteria and can be exploited in development of biotherapeutics. Other technologies, such as development of human gut models, bacterial screening, and delivery formulations eg, microencapsulated probiotics, may contribute significantly in the near future. Therefore, the human gut microbiota is a legitimate therapeutic target to treat and/or prevent various diseases. Development of a clear understanding of the technologies needed to exploit the gut microbiota is urgently required.Keywords: gut microbiota, human

  6. Design and Engineering of Deimmunized Biotherapeutics

    Science.gov (United States)

    Griswold, Karl E.; Bailey-Kellogg, Chris

    2016-01-01

    Therapeutic proteins are powerful next-generation drugs able to effectively treat diverse and devastating diseases, but the development and use of biotherapeutics entails unique challenges and risks. In particular, protein drugs are subject to immune surveillance in the human body, and ensuing antidrug immune responses can cause a wide range of problems including altered pharmacokinetics, loss of efficacy, and even life-threating complications. Here we review recent progress in technologies for engineering deimmunized biotherapeutics, placing particular emphasis on deletion of immunogenic antibody and T cell epitopes via experimentally or computationally guided mutagenesis. PMID:27322891

  7. Quantitative intracellular flux modeling and applications in biotherapeutic development and production using CHO cell cultures.

    Science.gov (United States)

    Huang, Zhuangrong; Lee, Dong-Yup; Yoon, Seongkyu

    2017-12-01

    Chinese hamster ovary (CHO) cells have been widely used for producing many recombinant therapeutic proteins. Constraint-based modeling, such as flux balance analysis (FBA) and metabolic flux analysis (MFA), has been developing rapidly for the quantification of intracellular metabolic flux distribution at a systematic level. Such methods would produce detailed maps of flows through metabolic networks, which contribute significantly to better understanding of metabolism in cells. Although these approaches have been extensively established in microbial systems, their application to mammalian cells is sparse. This review brings together the recent development of constraint-based models and their applications in CHO cells. The further development of constraint-based modeling approaches driven by multi-omics datasets is discussed, and a framework of potential modeling application in cell culture engineering is proposed. Improved cell culture system understanding will enable robust developments in cell line and bioprocess engineering thus accelerating consistent process quality control in biopharmaceutical manufacturing. © 2017 Wiley Periodicals, Inc.

  8. Leveraging a CHO cell line toolkit to accelerate biotherapeutics into the clinic.

    Science.gov (United States)

    Wright, Chapman; Alves, Christina; Kshirsagar, Rashmi; Pieracci, John; Estes, Scott

    2017-11-01

    The Biogen upstream platform is capable of delivering equivalent quality material throughout the cell line generation process. This allows us to rapidly deliver high-quality biopharmaceuticals to patients with unmet medical needs. The drive to reduce time-to-market led the cell engineering group to develop an expression system that can enable this strategy. We have developed a clonal Chinese Hamster Ovary (CHO) host cell line that can routinely produce consistent antibody material at high titers throughout the cell line generation process. This host line enables faster delivery of early phase material through use of the highly productive stable pool or a mixture of high performance clones. Due to unique characteristics of this cell line, the product quality of material from early cell populations is very comparable to material from the final clones. This lends itself to a "fast-to-tox" strategy whereby toxicology studies can be performed with representative material from an earlier cell population, thus accelerating the clinical timelines. Our new clonal host offers robust and consistent performance that enables a highly productive, flexible process and faster preclinical timelines. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1468-1475, 2017. © 2017 American Institute of Chemical Engineers.

  9. Analysis of host-cell proteins in biotherapeutic proteins by LC/MS approaches.

    Science.gov (United States)

    Doneanu, Catalin E; Chen, Weibin

    2014-01-01

    A generic method for the identification and quantification of host-cell proteins (HCPs) in protein biopharmaceuticals is described. Therapeutic proteins and HCPs were converted to complex peptide mixtures following tryptic digestion. Comprehensive peptide separations were performed using online two-dimensional capillary liquid chromatography-(LC) involving high-pH reversed phase (RP)/low-pH RP separations. We applied this method to the analysis of HCP impurities in monoclonal antibody (mAb) preparations.

  10. A novel sugar analog enhances sialic acid production and biotherapeutic sialylation in CHO cells.

    Science.gov (United States)

    Yin, Bojiao; Wang, Qiong; Chung, Cheng-Yu; Bhattacharya, Rahul; Ren, Xiaozhi; Tang, Juechun; Yarema, Kevin J; Betenbaugh, Michael J

    2017-08-01

    A desirable feature of many therapeutic glycoprotein production processes is to maximize the final sialic acid content. In this study, the effect of applying a novel chemical analog of the sialic acid precursor N-acetylmannosamine (ManNAc) on the sialic acid content of cellular proteins and a model recombinant glycoprotein, erythropoietin (EPO), was investigated in CHO-K1 cells. By introducing the 1,3,4-O-Bu 3 ManNAc analog at 200-300 µM into cell culture media, the intracellular sialic acid content of EPO-expressing cells increased ∼8-fold over untreated controls while the level of cellular sialylated glycoconjugates increased significantly as well. For example, addition of 200-300 µM 1,3,4-O-Bu 3 ManNAc resulted in >40% increase in final sialic acid content of recombinant EPO, while natural ManNAc at ∼100 times higher concentration of 20 mM produced a less profound change in EPO sialylation. Collectively, these results indicate that butyrate-derivatization of ManNAc improves the capacity of cells to incorporate exogenous ManNAc into the sialic acid biosynthetic pathway and thereby increase sialylation of recombinant EPO and other glycoproteins. This study establishes 1,3,4-O-Bu 3 ManNAc as a novel chemical supplement to improve glycoprotein quality and sialylation levels at concentrations orders of magnitude lower than alternative approaches. Biotechnol. Bioeng. 2017;114: 1899-1902. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  11. Bacterial Cell Mechanics.

    Science.gov (United States)

    Auer, George K; Weibel, Douglas B

    2017-07-25

    Cellular mechanical properties play an integral role in bacterial survival and adaptation. Historically, the bacterial cell wall and, in particular, the layer of polymeric material called the peptidoglycan were the elements to which cell mechanics could be primarily attributed. Disrupting the biochemical machinery that assembles the peptidoglycan (e.g., using the β-lactam family of antibiotics) alters the structure of this material, leads to mechanical defects, and results in cell lysis. Decades after the discovery of peptidoglycan-synthesizing enzymes, the mechanisms that underlie their positioning and regulation are still not entirely understood. In addition, recent evidence suggests a diverse group of other biochemical elements influence bacterial cell mechanics, may be regulated by new cellular mechanisms, and may be triggered in different environmental contexts to enable cell adaptation and survival. This review summarizes the contributions that different biomolecular components of the cell wall (e.g., lipopolysaccharides, wall and lipoteichoic acids, lipid bilayers, peptidoglycan, and proteins) make to Gram-negative and Gram-positive bacterial cell mechanics. We discuss the contribution of individual proteins and macromolecular complexes in cell mechanics and the tools that make it possible to quantitatively decipher the biochemical machinery that contributes to bacterial cell mechanics. Advances in this area may provide insight into new biology and influence the development of antibacterial chemotherapies.

  12. Bacterial Cell Wall Components

    Science.gov (United States)

    Ginsberg, Cynthia; Brown, Stephanie; Walker, Suzanne

    Bacterial cell-surface polysaccharides cells are surrounded by a variety of cell-surface structures that allow them to thrive in extreme environments. Components of the cell envelope and extracellular matrix are responsible for providing the cells with structural support, mediating intercellular communication, allowing the cells to move or to adhere to surfaces, protecting the cells from attack by antibiotics or the immune system, and facilitating the uptake of nutrients. Some of the most important cell wall components are polysaccharide structures. This review discusses the occurrence, structure, function, and biosynthesis of the most prevalent bacterial cell surface polysaccharides: peptidoglycan, lipopolysaccharide, arabinogalactan, and lipoarabinomannan, and capsular and extracellular polysaccharides. The roles of these polysaccharides in medicine, both as drug targets and as therapeutic agents, are also described.

  13. Superparamagnetic Iron Oxide Nanoparticle-Based Delivery Systems for Biotherapeutics

    Science.gov (United States)

    Mok, Hyejung; Zhang, Miqin

    2014-01-01

    Introduction Superparamagnetic iron oxide nanoparticle (SPION)-based carrier systems have many advantages over other nanoparticle-based systems. They are biocompatible, biodegradable, facilely tunable, and superparamagnetic and thus controllable by an external magnetic field. These attributes enable their broad biomedical applications. In particular, magnetically-driven carriers are drawing considerable interest as an emerging therapeutic delivery system because of their superior delivery efficiency. Area covered This article reviews the recent advances in use of SPION-based carrier systems to improve the delivery efficiency and target specificity of biotherapeutics. We examine various formulations of SPION-based delivery systems, including SPION micelles, clusters, hydrogels, liposomes, and micro/nanospheres, as well as their specific applications in delivery of biotherapeutics. Expert opinion Recently, biotherapeutics including therapeutic cells, proteins and genes have been studied as alternative treatments to various diseases. Despite the advantages of high target specificity and low adverse effects, clinical translation of biotherapeutics has been hindered by the poor stability and low delivery efficiency compared to chemical drugs. Accordingly, biotherapeutic delivery systems that can overcome these limitations are actively pursued. SPION-based materials can be ideal candidates for developing such delivery systems because of their excellent biocompatibility and superparamagnetism that enables long-term accumulation/retention at target sites by utilization of a suitable magnet. In addition, synthesis technologies for production of finely-tuned, homogeneous SPIONs have been well developed, which may promise their rapid clinical translation. PMID:23199200

  14. Biosensors of bacterial cells.

    Science.gov (United States)

    Burlage, Robert S; Tillmann, Joshua

    2017-07-01

    Biosensors are devices which utilize both an electrical component (transducer) and a biological component to study an environment. They are typically used to examine biological structures, organisms and processes. The field of biosensors has now become so large and varied that the technology can often seem impenetrable. Yet the principles which underlie the technology are uncomplicated, even if the details of the mechanisms are elusive. In this review we confine our analysis to relatively current advancements in biosensors for the detection of whole bacterial cells. This includes biosensors which rely on an added labeled component and biosensors which do not have a labeled component and instead detect the binding event or bound structure on the transducer. Methods to concentrate the bacteria prior to biosensor analysis are also described. The variety of biosensor types and their actual and potential uses are described. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Biotherapeutic effects of probiotic bacteria on candidiasis in immunodeficient mice.

    Science.gov (United States)

    Wagner, R D; Pierson, C; Warner, T; Dohnalek, M; Farmer, J; Roberts, L; Hilty, M; Balish, E

    1997-10-01

    Four species of probiotic bacteria were assessed for their capacities to protect athymic bg/bg-nu/nu and euthymic bg/bg-nu/+ mice from mucosal and systemic candidiasis. Each bacterial species and Candida albicans colonized the gastrointestinal tracts of both strains of mice. The presence of probiotic bacteria (Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus casei GG, or Bifidobacterium animalis) in the gastrointestinal tracts prolonged the survival of adult and neonatal bg/bg-nu/nu mice compared to that of isogenic mice colonized with C. albicans alone. The incidence of systemic candidiasis in bg/bg-nu/nu mice was significantly reduced by each of the four probiotic bacterial species. The numbers of C. albicans present in the alimentary tracts of euthymic bg/bg-nu/+ mice were significantly reduced by L. casei GG and B. animalis. None of the probiotic bacteria species completely prevented mucosal candidiasis, but B. animalis reduced its incidence and severity. Probiotic bacteria also modulated antibody- and cell-mediated immune responses to C. albicans. The prolonged survival of mice, decreased severity of mucosal and systemic candidiasis, modulation of immune responses, decreased number of C. albicans in the alimentary tract, and reduced numbers of orogastric infections demonstrated not only that probiotic bacteria have biotherapeutic potential for prophylaxis against and therapy of this fungal disease but also that probiotic bacteria protect mice from candidiasis by a variety of immunologic (thymic and extrathymic) and nonimmunologic mechanisms in this model.

  16. Application of CFD in Bioprocessing: Separation of mammalian cells using disc stack centrifuge during production of biotherapeutics.

    Science.gov (United States)

    Shekhawat, Lalita Kanwar; Sarkar, Jayati; Gupta, Rachit; Hadpe, Sandeep; Rathore, Anurag S

    2018-02-10

    Centrifugation continues to be one of the most commonly used unit operations for achieving efficient harvest of the product from the mammalian cell culture broth during production of therapeutic monoclonal antibodies (mAbs). Since the mammalian cells are known to be shear sensitive, optimal performance of the centrifuge requires a balance between productivity and shear. In this study, Computational Fluid Dynamics (CFD) has been successfully used as a tool to facilitate efficient optimization. Multiphase Eulerian-Eulerian model coupled with Gidaspow drag model along with Eulerian-Eulerian k-ε mixture turbulence model have been used to quantify the complex hydrodynamics of the centrifuge and thus evaluate the turbulent stresses generated by the centrifugal forces. An empirical model has been developed by statistical analysis of experimentally observed cell lysis data as a function of turbulent stresses. An operating window that offers the optimal balance between high productivity, high separation efficiency, and low cell damage has been identified by use of CFD modeling. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Molecular characterization of gluten hydrolysing Bacillus sp. and their efficacy and biotherapeutic potential as probiotics using Caco-2 cell line.

    Science.gov (United States)

    Rashmi, B S; Gayathri, D

    2017-09-01

    To isolate and characterize indigenous gluten hydrolysing bacteria from wheat sourdough and curd samples and further evaluation of their probiotic potentiality. Indigenous gluten hydrolysing isolates GS 33, GS 143, GS 181 and GS 188 were identified as Bacillus sp. by molecular-typing methods and studied extensively for their functional and probiotic attributes. All the tested isolates could survive at pH 2 and toxicity of 0·3% bile and also exhibited cell surface hydrophobicity and autoaggregation phenotype. The isolates were adhered strongly to Caco-2 cells and coaggregated with Escherichia coli MTCC 433 and Listeria monocytogenes MTCC 1143 preventing pathogen invasion into Caco-2 cells in vitro. In addition, the minimum inhibitory concentration of selected antibiotics for all the investigated gluten hydrolysing isolates was within the breakpoint values as recommended by the European Food Safety Authority. The indigenous high intensity gluten hydrolysing bacteria exhibited high resistance to gastric and pancreatic stress and possessed antibacterial, aggregation, adhesion and pathogen exclusion properties, and as a potential probiotics, either alone or in consortium would be useful in the development of gluten-free wheat foods. Exploring new indigenous gluten hydrolysing bacteria from wheat sourdough and curd samples would be beneficial in developing gluten-free wheat foods using potential indigenous probiotics. © 2017 The Society for Applied Microbiology.

  18. Bacterial cell culture

    OpenAIRE

    sprotocols

    2014-01-01

    ### Materials 1. Glass culture tubes with metal caps and labels - Growth medium, from media room or customized - Glass pipette tubes - Parafilm ### Equipment 1. Vortexer - Fireboy or Bunsen burner - Motorized pipette - Micropipettes and sterile tips ### Procedure For a typical liquid culture, use 5 ml of appropriate medium. The amount in each tube does not have to be exact if you are just trying to culture cells for their precious DNA. 1. Streak an a...

  19. Bacterial cells with improved tolerance to polyamines

    DEFF Research Database (Denmark)

    2017-01-01

    Provided are bacterial cells genetically modified to improve their tolerance to certain commodity chemicals, such as polyamines, and methods of preparing and using such bacterial cells for production of polyamines and other compounds....

  20. Bacterial cells with improved tolerance to polyols

    DEFF Research Database (Denmark)

    2017-01-01

    The present invention relates to bacterial cells genetically modified to improve their tolerance to certain commodity chemicals, such as diols and other polyols, and to methods of preparing and using such bacterial cells for production of polyols and other compounds....

  1. Microencapsulation for the Therapeutic Delivery of Drugs, Live Mammalian and Bacterial Cells, and Other Biopharmaceutics: Current Status and Future Directions

    Directory of Open Access Journals (Sweden)

    Catherine Tomaro-Duchesneau

    2013-01-01

    Full Text Available Microencapsulation is a technology that has shown significant promise in biotherapeutics, and other applications. It has been proven useful in the immobilization of drugs, live mammalian and bacterial cells and other cells, and other biopharmaceutics molecules, as it can provide material structuration, protection of the enclosed product, and controlled release of the encapsulated contents, all of which can ensure efficient and safe therapeutic effects. This paper is a comprehensive review of microencapsulation and its latest developments in the field. It provides a comprehensive overview of the technology and primary goals of microencapsulation and discusses various processes and techniques involved in microencapsulation including physical, chemical, physicochemical, and other methods involved. It also summarizes the state-of-the-art successes of microencapsulation, specifically with regard to the encapsulation of microorganisms, mammalian cells, drugs, and other biopharmaceutics in various diseases. The limitations and future directions of microencapsulation technologies are also discussed.

  2. Microencapsulation for the Therapeutic Delivery of Drugs, Live Mammalian and Bacterial Cells, and Other Biopharmaceutics: Current Status and Future Directions

    Science.gov (United States)

    Saha, Shyamali; Malhotra, Meenakshi; Kahouli, Imen; Prakash, Satya

    2013-01-01

    Microencapsulation is a technology that has shown significant promise in biotherapeutics, and other applications. It has been proven useful in the immobilization of drugs, live mammalian and bacterial cells and other cells, and other biopharmaceutics molecules, as it can provide material structuration, protection of the enclosed product, and controlled release of the encapsulated contents, all of which can ensure efficient and safe therapeutic effects. This paper is a comprehensive review of microencapsulation and its latest developments in the field. It provides a comprehensive overview of the technology and primary goals of microencapsulation and discusses various processes and techniques involved in microencapsulation including physical, chemical, physicochemical, and other methods involved. It also summarizes the state-of-the-art successes of microencapsulation, specifically with regard to the encapsulation of microorganisms, mammalian cells, drugs, and other biopharmaceutics in various diseases. The limitations and future directions of microencapsulation technologies are also discussed. PMID:26555963

  3. Immunogenicity of Biotherapeutics: Causes and Association with Posttranslational Modifications

    Directory of Open Access Journals (Sweden)

    Anshu Kuriakose

    2016-01-01

    Full Text Available Today, potential immunogenicity can be better evaluated during the drug development process, and we have rational approaches to manage the clinical consequences of immunogenicity. The focus of the scientific community should be on developing sensitive diagnostics that can predict immunogenicity-mediated adverse events in the small fraction of subjects that develop clinically relevant anti-drug antibodies. Here, we discuss the causes of immunogenicity which could be product-related (inherent property of the product or might be picked up during the manufacturing process, patient-related (genetic profile or eating habits, or linked to the route of administration. We describe various posttranslational modifications (PTMs and how they may influence immunogenicity. Over the last three decades, we have significantly improved our understanding about the types of PTMs of biotherapeutic proteins and their association with immunogenicity. It is also now clear that all PTMs do not lead to clinical immunogenicity. We also discuss the mechanisms of immunogenicity (which include T cell-dependent and T cell-independent responses and immunological tolerance. We further elaborate on the management of immunogenicity in preclinical and clinical setting and the unique challenges raised by biosimilars, which may have different immunogenic potential from their parent biotherapeutics.

  4. 77 FR 9947 - Guidance for Industry: Early Clinical Trials With Live Biotherapeutic Products: Chemistry...

    Science.gov (United States)

    2012-02-21

    ...] Guidance for Industry: Early Clinical Trials With Live Biotherapeutic Products: Chemistry, Manufacturing... ``Guidance for Industry: Early Clinical Trials With Live Biotherapeutic Products: Chemistry, Manufacturing... for Industry: Early Clinical Trials With Live Biotherapeutic Products: Chemistry, Manufacturing, and...

  5. Pre-existing Antibody: Biotherapeutic Modality-Based Review

    OpenAIRE

    Gorovits, Boris; Clements-Egan, Adrienne; Birchler, Mary; Liang, Meina; Myler, Heather; Peng, Kun; Purushothama, Shobha; Rajadhyaksha, Manoj; Salazar-Fontana, Laura; Sung, Crystal; Xue, Li

    2016-01-01

    Pre-existing antibodies to biotherapeutic drugs have been detected in drug-naïve subjects for a variety of biotherapeutic modalities. Pre-existing antibodies are immunoglobulins that are either specific or cross-reacting with a protein or glycan epitopes on a biotherapeutic compound. Although the exact cause for pre-existing antibodies is often unknown, environmental exposures to non-human proteins, glycans, and structurally similar products are frequently proposed as factors. Clinical conseq...

  6. Cell cycle regulation by the bacterial nucleoid

    OpenAIRE

    Adams, David William; Wu, Ling Juan; Errington, Jeff

    2014-01-01

    Division site selection presents a fundamental challenge to all organisms. Bacterial cells are small and the chromosome (nucleoid) often fills most of the cell volume. Thus, in order to maximise fitness and avoid damaging the genetic material, cell division must be tightly co-ordinated with chromosome replication and segregation. To achieve this, bacteria employ a number of different mechanisms to regulate division site selection. One such mechanism, termed nucleoid occlusion, allows the nucl...

  7. Bacterial Networks in Cells and Communities.

    Science.gov (United States)

    Sourjik, Victor; Vorholt, Julia A

    2015-11-20

    Research on the bacterial regulatory networks is currently experiencing a true revival, driven by advances in methodology and by emergence of novel concepts. The biannual conference Bacterial Networks (BacNet15) held in May 2015, in Sant Feliu de Guíxols, Spain, covered progress in the studies of regulatory networks that control bacterial physiology, cell biology, stress responses, metabolism, collective behavior and evolution. It demonstrated how interdisciplinary approaches that combine molecular biology and biochemistry with the latest microscopy developments, whole cell (-omics) approaches and mathematical modeling can help understand design principles relevant in microbiology. It further showed how current biotechnology and medical microbiology could profit from our knowledge of and ability to engineer regulatory networks of bacteria. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Bacterial Cell Wall Growth, Shape and Division

    NARCIS (Netherlands)

    Derouaux, A.; Terrak, M.; den Blaauwen, T.; Vollmer, W.; Remaut, H.; Fronzes, R.

    2014-01-01

    The shape of a bacterial cell is maintained by its peptidoglycan sacculus that completely surrounds the cytoplasmic membrane. During growth the sacculus is enlarged by peptidoglycan synthesis complexes that are controlled by components linked to the cytoskeleton and, in Gram-negative bacteria, by

  9. Electrochemical characterization of the bacterial cell surface

    NARCIS (Netherlands)

    Wal, van der A.

    1996-01-01


    Bacterial cells are ubiquitous in natural environments and also play important roles in domestic and industrial processes. They are found either suspended in the aqueous phase or attached to solid particles. The adhesion behaviour of bacteria is influenced by the physico-chemical

  10. Establishment of a tobacco BY2 cell line devoid of plant-specific xylose and fucose as a platform for the production of biotherapeutic proteins.

    Science.gov (United States)

    Hanania, Uri; Ariel, Tami; Tekoah, Yoram; Fux, Liat; Sheva, Maor; Gubbay, Yehuda; Weiss, Mara; Oz, Dina; Azulay, Yaniv; Turbovski, Albina; Forster, Yehava; Shaaltiel, Yoseph

    2017-09-01

    Plant-produced glycoproteins contain N-linked glycans with plant-specific residues of β(1,2)-xylose and core α(1,3)-fucose, which do not exist in mammalian-derived proteins. Although our experience with two enzymes that are used for enzyme replacement therapy does not indicate that the plant sugar residues have deleterious effects, we made a conscious decision to eliminate these moieties from plant-expressed proteins. We knocked out the β(1,2)-xylosyltranferase (XylT) and the α(1,3)-fucosyltransferase (FucT) genes, using CRISPR/Cas9 genome editing, in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. In total, we knocked out 14 loci. The knocked-out lines were stable, viable and exhibited a typical BY2 growing rate. Glycan analysis of the endogenous proteins of these lines exhibited N-linked glycans lacking β(1,2)-xylose and/or α(1,3)-fucose. The knocked-out lines were further transformed successfully with recombinant DNaseI. The expression level and the activity of the recombinant protein were similar to that of the protein produced in the wild-type BY2 cells. The recombinant DNaseI was shown to be totally free from any xylose and/or fucose residues. The glyco-engineered BY2 lines provide a valuable platform for producing potent biopharmaceutical products. Furthermore, these results demonstrate the power of the CRISPR/Cas9 technology for multiplex gene editing in BY2 cells. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  11. Towards regulation of similar biotherapeutic products: Thailand's perspective.

    Science.gov (United States)

    Thanaphollert, Prapassorn; Tungsanga, Kriang

    2011-09-01

    The implementation of universal health coverage scheme in Thailand allows quality, equitable and accessible health care for all. Patients with life threatening and chronic diseases can get access to biotherapeutic products to treat their ailments. This triggered a major impact on the need for specific guidelines in evaluation of similar biotherapeutic products in order to standardize the regulatory pathway to license this class of products ensuring that the products meet acceptable levels of quality, safety and efficacy. The development of similar biotherapeutic products (SBP) should be considered to ensure therapeutic equivalence of biotherapeutics products at more affordable prices. This will lead to greater ease and speed of approval and assurance of the quality, safety and efficacy of these products. Therefore, we report herein the SBP situation in Thailand. Copyright © 2011. Published by Elsevier Ltd.

  12. Selective adsorption of bacterial cells onto zeolites.

    Science.gov (United States)

    Kubota, Munehiro; Nakabayashi, Tadashi; Matsumoto, Yuki; Shiomi, Tohru; Yamada, Yusuke; Ino, Keita; Yamanokuchi, Hiroyuki; Matsui, Masayoshi; Tsunoda, Tatsuo; Mizukami, Fujio; Sakaguchi, Kengo

    2008-06-15

    Zeolites adsorb microbial cells on their surfaces and selective adsorption for specific microorganisms was seen with certain zeolites. Tests for the adsorption ability of zeolites were conducted using various established microbial cell lines. Specific cell lines were shown to selectively absorb to certain zeolites, species to species. In order to understand the selectivity of adsorption, we tested adsorption under various pH conditions and determined the zeta-potentials of zeolites and cells. The adsorption of some cell lines depended on the pH, and some microorganisms were preferentially adsorbed at acidic pH. The values of zeta-potentials were used for calculating the electric double layer interaction energy between zeolites and microbial cells. There was a correlation between the experimental adsorption results and the interaction energy. Moreover, we evaluated the surface hydrophobicity of bacterial cells by using the microbial adherence to hydrocarbon (MATH) assay. In addition, we also applied this method for zeolites to quantify relative surface hydrophobicity. As a result, we found a correlation between the adsorption results and the hydrophobicity of bacterial cells and zeolites. These results suggested that adsorption could be explained mainly by electric double layer interactions and hydrophobic interactions. Finally, by using the zeolites Na-BEA and H-Y, we succeeded in clearly separating three representative microbes from a mixture of Escherichia coli, Bacillus subtilis and Staphylococcus aureus. Zeolites could adsorb each of the bacterial cell species with high selectivity even from a mixed suspension. Zeolites can therefore be used as effective carrier materials to provide an easy, rapid and accurate method for cell separation.

  13. Bacterial cells with improved tolerance to isobutyric acid

    DEFF Research Database (Denmark)

    2017-01-01

    Bacterial cells genetically modified to improve their tolerance to certain commodity chemicals, such as isobutyric acid and related compounds, and methods of preparing and using such bacterial cells for production of isobutyric acid and related compounds....

  14. Biosensors for Whole-Cell Bacterial Detection

    Science.gov (United States)

    Rushworth, Jo V.; Hirst, Natalie A.; Millner, Paul A.

    2014-01-01

    SUMMARY Bacterial pathogens are important targets for detection and identification in medicine, food safety, public health, and security. Bacterial infection is a common cause of morbidity and mortality worldwide. In spite of the availability of antibiotics, these infections are often misdiagnosed or there is an unacceptable delay in diagnosis. Current methods of bacterial detection rely upon laboratory-based techniques such as cell culture, microscopic analysis, and biochemical assays. These procedures are time-consuming and costly and require specialist equipment and trained users. Portable stand-alone biosensors can facilitate rapid detection and diagnosis at the point of care. Biosensors will be particularly useful where a clear diagnosis informs treatment, in critical illness (e.g., meningitis) or to prevent further disease spread (e.g., in case of food-borne pathogens or sexually transmitted diseases). Detection of bacteria is also becoming increasingly important in antibioterrorism measures (e.g., anthrax detection). In this review, we discuss recent progress in the use of biosensors for the detection of whole bacterial cells for sensitive and earlier identification of bacteria without the need for sample processing. There is a particular focus on electrochemical biosensors, especially impedance-based systems, as these present key advantages in terms of ease of miniaturization, lack of reagents, sensitivity, and low cost. PMID:24982325

  15. Measuring bacterial cells size with AFM

    Directory of Open Access Journals (Sweden)

    Denise Osiro

    2012-03-01

    Full Text Available Atomic Force Microscopy (AFM can be used to obtain high-resolution topographical images of bacteria revealing surface details and cell integrity. During scanning however, the interactions between the AFM probe and the membrane results in distortion of the images. Such distortions or artifacts are the result of geometrical effects related to bacterial cell height, specimen curvature and the AFM probe geometry. The most common artifact in imaging is surface broadening, what can lead to errors in bacterial sizing. Several methods of correction have been proposed to compensate for these artifacts and in this study we describe a simple geometric model for the interaction between the tip (a pyramidal shaped AFM probe and the bacterium (Escherichia coli JM-109 strain to minimize the enlarging effect. Approaches to bacteria immobilization and examples of AFM images analysis are also described.

  16. Elastic Deformations During Bacterial Cell Growth

    Science.gov (United States)

    Huang, K. C.

    2010-03-01

    The wide variety of shapes and sizes found in bacterial species is almost universally defined by the cell wall, which is a cross-linked network of the material peptidoglycan. In recent years, cell shape has been shown to play a critical role in regulating many important biological functions including attachment, dispersal, motility, polar differentiation, predation, and cellular differentiation. In previous work, we have shown that the spatial organization of the peptidoglycan network can change the mechanical equilibrium of the cell wall and result in changes in cell shape. However, experimental data on the mechanical properties of peptidoglycan is currently limited. Here, we describe a straightforward, inexpensive approach for extracting the mechanical properties of bacterial cells in gels of user-defined stiffness, using only optical microscopy to match growth kinetics to the predictions of a continuum model of cell growth. Using this simple yet general methodology, we have measured the Young's modulus for bacteria ranging across a wide variety of shapes, sizes, and cell wall thicknesses, and our method can easily be extended to other commonly studied bacteria. This method makes it possible to rapidly determine how changes in genotype and biochemistry affect the mechanical properties of the cell wall, and may be particularly relevant for studying the relationship between cell shape and structure, the genetic and molecular control of the mechanical properties of the cell wall, and the identification of antibiotics and other small molecules that affect and specifically modify the mechanical properties of the cell wall. Our work also suggests that bacteria may utilize peptidoglycan synthesis to transduce mechanosensory signals from local environment.

  17. Cell cycle regulation by the bacterial nucleoid.

    Science.gov (United States)

    Adams, David William; Wu, Ling Juan; Errington, Jeff

    2014-12-01

    Division site selection presents a fundamental challenge to all organisms. Bacterial cells are small and the chromosome (nucleoid) often fills most of the cell volume. Thus, in order to maximise fitness and avoid damaging the genetic material, cell division must be tightly co-ordinated with chromosome replication and segregation. To achieve this, bacteria employ a number of different mechanisms to regulate division site selection. One such mechanism, termed nucleoid occlusion, allows the nucleoid to protect itself by acting as a template for nucleoid occlusion factors, which prevent Z-ring assembly over the DNA. These factors are sequence-specific DNA-binding proteins that exploit the precise organisation of the nucleoid, allowing them to act as both spatial and temporal regulators of bacterial cell division. The identification of proteins responsible for this process has provided a molecular understanding of nucleoid occlusion but it has also prompted the realisation that substantial levels of redundancy exist between the diverse systems that bacteria employ to ensure that division occurs in the right place, at the right time.

  18. Pre-existing Antibody: Biotherapeutic Modality-Based Review.

    Science.gov (United States)

    Gorovits, Boris; Clements-Egan, Adrienne; Birchler, Mary; Liang, Meina; Myler, Heather; Peng, Kun; Purushothama, Shobha; Rajadhyaksha, Manoj; Salazar-Fontana, Laura; Sung, Crystal; Xue, Li

    2016-03-01

    Pre-existing antibodies to biotherapeutic drugs have been detected in drug-naïve subjects for a variety of biotherapeutic modalities. Pre-existing antibodies are immunoglobulins that are either specific or cross-reacting with a protein or glycan epitopes on a biotherapeutic compound. Although the exact cause for pre-existing antibodies is often unknown, environmental exposures to non-human proteins, glycans, and structurally similar products are frequently proposed as factors. Clinical consequences of the pre-existing antibodies vary from an adverse effect on patient safety to no impact at all and remain highly dependent on the biotherapeutic drug modality and therapeutic indication. As such, pre-existing antibodies are viewed as an immunogenicity risk factor requiring a careful evaluation. Herein, the relationships between biotherapeutic modalities to the nature, prevalence, and clinical consequences of pre-existing antibodies are reviewed. Initial evidence for pre-existing antibody is often identified during anti-drug antibody (ADA) assay development. Other interfering factors known to cause false ADA positive signal, including circulating multimeric drug target, rheumatoid factors, and heterophilic antibodies, are discussed.

  19. Dynamic Viscoelasticity of Individual Bacterial Cells

    Science.gov (United States)

    Vadillo-Rodriguez, Virginia; Dutcher, John

    2009-03-01

    We have used an AFM-based approach to probe the mechanical properties of single bacterial cells (gram-negative Escherichia coli K12) by applying a constant compressive force to the cell under fluid conditions while measuring the time-dependent displacement (creep) of a colloidal AFM tip due to the viscoelastic properties of the cell. We observed that the cells exhibited a viscoelastic solid-like behavior with retarded elasticity, i.e. both an instantaneous and a delayed elastic deformation, which is well described by a three-parameter mechanical model. Using the best fit parameter values, we have calculated the dynamic viscoelastic behavior of the cells over a wide range of frequencies based on a numerical time-frequency transform technique and we have compared the calculated behavior with that measured experimentally. Comparison of the results obtained for E. coli with previously reported data on the mechanical properties of others gram-negative cells and their isolated surface layers suggests that the elastic component of the cell viscoelastic response is dominated by the properties of the peptidoglycan layer, whereas the viscous component likely arises from the liquid-like character of the cell membranes.

  20. One Bacterial Cell, One Complete Genome

    Energy Technology Data Exchange (ETDEWEB)

    Woyke, Tanja; Tighe, Damon; Mavrommatis, Konstantinos; Clum, Alicia; Copeland, Alex; Schackwitz, Wendy; Lapidus, Alla; Wu, Dongying; McCutcheon, John P.; McDonald, Bradon R.; Moran, Nancy A.; Bristow, James; Cheng, Jan-Fang

    2010-04-26

    While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200?900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA). Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs), indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.

  1. One bacterial cell, one complete genome.

    Directory of Open Access Journals (Sweden)

    Tanja Woyke

    2010-04-01

    Full Text Available While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200-900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA. Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs, indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.

  2. Probing bacterial adhesion at the single-cell level

    DEFF Research Database (Denmark)

    Zeng, Guanghong; Müller, Torsten; Meyer, Rikke Louise

    Bacteria initiate attachment to surfaces with the aid of different extracellular proteins and polymeric adhesins. To quantitatively analyse the cell-cell and cell-surface interactions provided by bacterial adhesins, it is essential to go down to single cell level where cell-to-cell variation can...... be considered. We have developed a simple and versatile method to make single-cell bacterial probes for measuring single cell adhesion by force spectroscopy using atomic force microscopy (AFM). A single-cell probe was readily made by picking up a bacterial cell from a glass surface by approaching a tipless AFM...... cantilever coated with the commercial cell adhesive CellTakTM. We applied the method to study adhesion of living cells to abiotic surfaces at the single-cell level. Immobilisation of single bacterial cells to the cantilever was stable for several hours, and viability was confirmed by Live/Dead staining...

  3. Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation.

    Science.gov (United States)

    Ghaderi, Darius; Zhang, Mai; Hurtado-Ziola, Nancy; Varki, Ajit

    2012-01-01

    One of the fastest growing fields in the pharmaceutical industry is the market for therapeutic glycoproteins. Today, these molecules play a major role in the treatment of various diseases, and include several protein classes, i.e., clotting factors, hormones, cytokines, antisera, enzymes, enzyme inhibitors, Ig-Fc-Fusion proteins, and monoclonal antibodies. Optimal glycosylation is critical for therapeutic glycoproteins, as glycans can influence their yield, immunogenicity and efficacy, which impact the costs and success of such treatments. While several mammalian cell expression systems currently used can produce therapeutic glycoproteins that are mostly decorated with human-like glycans, they can differ from human glycans by presenting two structures at the terminal and therefore most exposed position. First, natural human N-glycans are lacking the terminal Gal 1-3Gal (alpha-Gal) modification; and second, they do not contain the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc). All humans spontaneously express antibodies against both of these glycan structures, risking increased immunogenicity of biotherapeutics carrying such non-human glycan epitopes. However, in striking contrast to the alpha-Gal epitope, exogenous Neu5Gc can be metabolically incorporated into human cells and presented on expressed glycoproteins in several possible epitopes. Recent work has demonstrated that this non-human sialic acid is found in widely varying amounts on biotherapeutic glycoproteins approved for treatment of various medical conditions. Neu5Gc on glycans of these medical agents likely originates from the production process involving the non-human mammalian cell lines and/or the addition of animal-derived tissue culture supplements. Further studies are needed to fully understand the impact of Neu5Gc in biotherapeutic agents. Similar concerns apply to human cells prepared for allo- or auto-transplantation, that have been grown in animal-derived tissue culture supplements.

  4. Biotherapeutic bioanalysis: a multi-indication case study review.

    Science.gov (United States)

    Myler, Heather A; Given, Allison; Kolz, Karen; Mora, Johanna R; Hristopoulos, George

    2011-03-01

    A thorough understanding of the structure and biology of a biotherapeutic is crucial to defining a suitable strategy for pharmacokinetic characterization in proof-of-concept disease models, toxicology species as well as the healthy and disease indication patient populations. This manuscript summarizes parameters that impact bioanalytical strategy for over 50 biotherapeutics indicated for the treatment of oncology, rheumatoid arthritis, allergy, multiple sclerosis, hematology, metabolism and infectious disease. We have addressed numerous therapeutic modalities including chimeric, humanized and fully human monoclonal antibodies, replacement proteins, peptides and fusion proteins, including polyethylene glycol and Fc fusions, as well as antibody-drug conjugates. With the rapid evolution of biotherapeutics over the last 20 years and the contraction of the pharmaceutical and biotechnology labor force, efficient workflow management becomes a crucial bioanalytical component. Thus, we have also addressed new technologies that have demonstrated either increased throughput or enhanced characterization, including Meso Scale Discovery, Gyrolab and affinity MS.

  5. Isoprenoid Biosynthesis Inhibitors Targeting Bacterial Cell Growth.

    Science.gov (United States)

    Desai, Janish; Wang, Yang; Wang, Ke; Malwal, Satish R; Oldfield, Eric

    2016-10-06

    We synthesized potential inhibitors of farnesyl diphosphate synthase (FPPS), undecaprenyl diphosphate synthase (UPPS), or undecaprenyl diphosphate phosphatase (UPPP), and tested them in bacterial cell growth and enzyme inhibition assays. The most active compounds were found to be bisphosphonates with electron-withdrawing aryl-alkyl side chains which inhibited the growth of Gram-negative bacteria (Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa) at ∼1-4 μg mL -1 levels. They were found to be potent inhibitors of FPPS; cell growth was partially "rescued" by the addition of farnesol or overexpression of FPPS, and there was synergistic activity with known isoprenoid biosynthesis pathway inhibitors. Lipophilic hydroxyalkyl phosphonic acids inhibited UPPS and UPPP at micromolar levels; they were active (∼2-6 μg mL -1 ) against Gram-positive but not Gram-negative organisms, and again exhibited synergistic activity with cell wall biosynthesis inhibitors, but only indifferent effects with other inhibitors. The results are of interest because they describe novel inhibitors of FPPS, UPPS, and UPPP with cell growth inhibitory activities as low as ∼1-2 μg mL -1 . © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Bioinspired Nanocarriers Designed to Enhance Intracellular Delivery of Biotherapeutics

    Science.gov (United States)

    2001-10-25

    therapeutic and vaccine development. Keywords - gene therapy, vaccine, bioinspired, biotherapeutic I. INTRODUCTION The efficacy of many protein and DNA...DNA, RNA and proteins . While these therapeutics have tremendous potential, effectively formulating and delivering them has also been a widely...intracellular trafficking that is inspired by biological polymers, i.e. proteins , that are involved in controlling vesicular trafficking pathways. For

  7. Bacterial cell curvature through mechanical control of cell growth

    DEFF Research Database (Denmark)

    Cabeen, M.; Charbon, Godefroid; Vollmer, W.

    2009-01-01

    The cytoskeleton is a key regulator of cell morphogenesis. Crescentin, a bacterial intermediate filament-like protein, is required for the curved shape of Caulobacter crescentus and localizes to the inner cell curvature. Here, we show that crescentin forms a single filamentous structure...... that collapses into a helix when detached from the cell membrane, suggesting that it is normally maintained in a stretched configuration. Crescentin causes an elongation rate gradient around the circumference of the sidewall, creating a longitudinal cell length differential and hence curvature. Such curvature...... can be produced by physical force alone when cells are grown in circular microchambers. Production of crescentin in Escherichia coli is sufficient to generate cell curvature. Our data argue for a model in which physical strain borne by the crescentin structure anisotropically alters the kinetics...

  8. Bacterial Vaginosis Bacterial and Epithelial Cell Adhesion Molecules

    Directory of Open Access Journals (Sweden)

    Şayeste Demirezen

    2016-05-01

    molecules. The most important adhesion molecules of epithelium are cadherins, fibronectins, Toll like receptors and carbohydrates. In bacteria, pilis, lypopolysaccaharide and biofilm have primary importance. In this review, the adhesion molecules are discussed in detail and their roles in formation of clue cell are clarified.

  9. Mapping the Pareto optimal design space for a functionally deimmunized biotherapeutic candidate.

    Science.gov (United States)

    Salvat, Regina S; Parker, Andrew S; Choi, Yoonjoo; Bailey-Kellogg, Chris; Griswold, Karl E

    2015-01-01

    The immunogenicity of biotherapeutics can bottleneck development pipelines and poses a barrier to widespread clinical application. As a result, there is a growing need for improved deimmunization technologies. We have recently described algorithms that simultaneously optimize proteins for both reduced T cell epitope content and high-level function. In silico analysis of this dual objective design space reveals that there is no single global optimum with respect to protein deimmunization. Instead, mutagenic epitope deletion yields a spectrum of designs that exhibit tradeoffs between immunogenic potential and molecular function. The leading edge of this design space is the Pareto frontier, i.e. the undominated variants for which no other single design exhibits better performance in both criteria. Here, the Pareto frontier of a therapeutic enzyme has been designed, constructed, and evaluated experimentally. Various measures of protein performance were found to map a functional sequence space that correlated well with computational predictions. These results represent the first systematic and rigorous assessment of the functional penalty that must be paid for pursuing progressively more deimmunized biotherapeutic candidates. Given this capacity to rapidly assess and design for tradeoffs between protein immunogenicity and functionality, these algorithms may prove useful in augmenting, accelerating, and de-risking experimental deimmunization efforts.

  10. Mapping the Pareto optimal design space for a functionally deimmunized biotherapeutic candidate.

    Directory of Open Access Journals (Sweden)

    Regina S Salvat

    2015-01-01

    Full Text Available The immunogenicity of biotherapeutics can bottleneck development pipelines and poses a barrier to widespread clinical application. As a result, there is a growing need for improved deimmunization technologies. We have recently described algorithms that simultaneously optimize proteins for both reduced T cell epitope content and high-level function. In silico analysis of this dual objective design space reveals that there is no single global optimum with respect to protein deimmunization. Instead, mutagenic epitope deletion yields a spectrum of designs that exhibit tradeoffs between immunogenic potential and molecular function. The leading edge of this design space is the Pareto frontier, i.e. the undominated variants for which no other single design exhibits better performance in both criteria. Here, the Pareto frontier of a therapeutic enzyme has been designed, constructed, and evaluated experimentally. Various measures of protein performance were found to map a functional sequence space that correlated well with computational predictions. These results represent the first systematic and rigorous assessment of the functional penalty that must be paid for pursuing progressively more deimmunized biotherapeutic candidates. Given this capacity to rapidly assess and design for tradeoffs between protein immunogenicity and functionality, these algorithms may prove useful in augmenting, accelerating, and de-risking experimental deimmunization efforts.

  11. Pre-Existing Biotherapeutic-Reactive Antibodies: Survey Results Within the American Association of Pharmaceutical Scientists

    OpenAIRE

    Xue, Li; Fiscella, Michele; Rajadhyaksha, Manoj; Goyal, Jaya; Holland, Claire; Gorovits, Boris; Morimoto, Alyssa

    2013-01-01

    The immunogenicity profile of a biotherapeutic is determined by a multitude of product and patient-related risk factors that can influence the observed incidence and clinical consequences of immunogenicity. Pre-existing antibodies, i.e., biotherapeutic-reactive antibodies present in samples from treatment-naïve subjects, have been commonly observed during immunogenicity assessments; however their relevance in terms of the safety and efficacy of a biotherapeutic is poorly understood. An Americ...

  12. Delivering natural products and biotherapeutics to improve drug efficacy.

    Science.gov (United States)

    Obeid, Mohammad A; Al Qaraghuli, Mohammed M; Alsaadi, Manal; Alzahrani, Abdullah R; Niwasabutra, Kanidta; Ferro, Valerie A

    2017-11-01

    Due to the increasing problem of drug resistance, new and improved medicines are required. Natural products and biotherapeutics offer a vast resource for new drugs; however, challenges, including the cost and time taken for traditional drug discovery processes and the subsequent lack of investment from the pharmaceutical industry, are associated with these areas. New techniques are producing compounds with appropriate activity at a faster rate. While the formulation of these combined with drug-delivery systems offers a promising approach for expanding the drug developments available to modern medicine. Here, various classes of drug-delivery systems are described and the advantages they bring to small molecule and biotherapeutic targeting are highlighted. This is an attractive approach to the pharmaceutical industry and the rising trend in research in this area is examined in brief. [Formula: see text].

  13. Bacterial Cell Surface Damage Due to Centrifugal Compaction

    NARCIS (Netherlands)

    Peterson, Brandon W.; Sharma, Prashant K.; van der Mei, Henny C.; Busscher, Henk J.

    Centrifugal damage has been known to alter bacterial cell surface properties and interior structures, including DNA. Very few studies exist on bacterial damage caused by centrifugation because of the difficulty in relating centrifugation speed and container geometry to the damage caused. Here, we

  14. Immersion Refractometry of Isolated Bacterial Cell Walls

    Science.gov (United States)

    Marquis, Robert E.

    1973-01-01

    Immersion-refractometric and light-scattering measurements were adapted to determinations of average refractive indices and physical compactness of isolated bacterial cell walls. The structures were immersed in solutions containing various concentrations of polymer molecules that cannot penetrate into wall pores, and then an estimate was made of the polymer concentration or the refractive index of the polymer solution in which light scattering was reduced to zero. Because each wall preparation was heterogeneous, the refractive index of the medium for zero light scattering had to be estimated by extrapolation. Refractive indices for walls suspended in bovine serum albumin solutions ranged from 1.348 for walls of the rod form of Arthrobacter crystallopoietes to 1.382 for walls of the teichoic acid deficient, 52A5 strain of Staphylococcus aureus. These indices were used to calculate approximate values for solids content per milliliter, and the calculated values agreed closely with those estimated from a knowledge of dextran-impermeable volumes per gram, dry weight, of the walls. When large molecules such as dextrans or serum albumin were used for immersion refractometry, the refractive indices obtained were for entire walls, including both wall polymers and wall water. When smaller molecules that can penetrate wall pores to various extents were used with Micrococcus lysodeikticus walls, the average, apparent refractive index of the structures increased as the molecular size of probing molecules was decreased. It was possible to obtain an estimate of 1.45 to 1.46 for the refractive index of wall polymers, predominantly peptidoglycans in this case, by extrapolating the curve for refractive index versus molecular radius to a value of 0.2 nm, the approximate radius of a water molecule. This relatively low value for polymer refractive index was interpreted as evidence in favor of the amorphous, elastic model of peptidoglycan structure and against the crystalline, rigid

  15. Photopolymerization Synthesis of Magnetic Nanoparticle Embedded Nanogels for Targeted Biotherapeutic Delivery

    Science.gov (United States)

    Denmark, Daniel J.

    Conventional therapeutic techniques treat the patient by delivering a biotherapeutic to the entire body rather than the target tissue. In the case of chemotherapy, the biotherapeutic is a drug that kills healthy and diseased cells indiscriminately which can lead to undesirable side effects. With targeted delivery, biotherapeutics can be delivered directly to the diseased tissue significantly reducing exposure to otherwise healthy tissue. Typical composite delivery devices are minimally composed of a stimuli responsive polymer, such as poly(N-isopropylacrylamide), allowing for triggered release when heated beyond approximately 32 °C, and magnetic nanoparticles which enable targeting as well as provide a mechanism for stimulus upon alternating magnetic field heating. Although more traditional methods, such as emulsion polymerization, have been used to realize these composite devices, the synthesis is problematic. Poisonous surfactants that are necessary to prevent agglomeration must be removed from the finished polymer, increasing the time and cost of the process. This study seeks to further explore non-toxic, biocompatible, non-residual, photochemical methods of creating stimuli responsive nanogels to advance the targeted biotherapeutic delivery field. Ultraviolet photopolymerization promises to be more efficient, while ensuring safety by using only biocompatible substances. The reactants selected for nanogel fabrication were N -isopropylacrylamide as monomer, methylene bisacrylamide as cross-linker, and Irgacure 2959 as ultraviolet photo-initiator. The superparamagnetic nanoparticles for encapsulation were approximately 10 nm in diameter and composed of magnetite to enable remote delivery and enhanced triggered release properties. Early investigations into the interactions of the polymer and nanoparticles employ a pioneering experimental setup, which allows for coincident turbidimetry and alternating magnetic field heating of an aqueous solution containing both

  16. New Application of Hyperspectral Imaging for Bacterial Cell Classification

    Science.gov (United States)

    Hyperspectral microscopy has shown potential as a method for rapid detection of foodborne pathogenic bacteria with spectral characteristics from bacterial cells. Hyperspectral microscope images (HMIs) are collected from broiler chicken isolates of Salmonella serotypes Enteritidis, Typhimurium, Infa...

  17. Osmotic Pressure, Bacterial Cell Walls, and Penicillin: A Demonstration.

    Science.gov (United States)

    Lennox, John E.

    1984-01-01

    An easily constructed apparatus that models the effect of penicillin on the structure of bacterial cells is described. Background information and procedures for using the apparatus during a classroom demonstration are included. (JN)

  18. Secukinumab, a novel anti–IL-17A antibody, shows low immunogenicity potential in human in vitro assays comparable to other marketed biotherapeutics with low clinical immunogenicity

    Science.gov (United States)

    Karle, Anette; Spindeldreher, Sebastian; Kolbinger, Frank

    2016-01-01

    ABSTRACT Secukinumab is a human monoclonal antibody that selectively targets interleukin-17A and has been demonstrated to be highly efficacious in the treatment of moderate to severe plaque psoriasis, starting at early time points, with a sustained effect and a favorable safety profile. Biotherapeutics—including monoclonal antibodies (mAbs)—can be immunogenic, leading to formation of anti-drug antibodies (ADAs) that can result in unwanted effects, including hypersensitivity reactions or compromised therapeutic efficacy. To gain insight into possible explanations for the clinically observed low immunogenicity of secukinumab, we evaluated its immunogenicity potential by applying 2 different in vitro assays: T-cell activation and major histocompatibility complex–associated peptide proteomics (MAPPs). For both assays, monocyte-derived dendritic cells (DCs) from healthy donors were exposed in vitro to biotherapeutic proteins. DCs naturally process proteins and present the derived peptides in the context of human leukocyte antigen (HLA)-class II. HLA-DR–associated biotherapeutic-derived peptides, representing potential T–cell epitopes, were identified in the MAPPs assay. In the T-cell assay, autologous CD4+ T cells were co-cultured with secukinumab-exposed DCs and T-cell activation was measured by proliferation and interleukin-2 secretion. In the MAPPs analysis and T-cell activation assays, secukinumab consistently showed relatively low numbers of potential T-cell epitopes and low T-cell response rates, respectively, comparable to other biotherapeutics with known low clinical immunogenicity. In contrast, biotherapeutics with elevated clinical immunogenicity rates showed increased numbers of potential T-cell epitopes and increased T-cell response rates in T-cell activation assays, indicating an approximate correlation between in vitro assay results and clinical immunogenicity incidence. PMID:26817498

  19. A gastrointestinal anti-infectious biotherapeutic agent: the heat-treated Lactobacillus LB.

    Science.gov (United States)

    Liévin-Le Moal, Vanessa

    2016-01-01

    Experimental in vitro and in vivo studies support the hypothesis that heat-treated, lyophilized Lactobacillus acidophilus LB cells and concentrated, neutralized spent culture medium conserve the variety of pharmacological, antimicrobial activities of the live probiotic strain against several infectious agents involved in well-established acute and persistent watery diarrhoea and gastritis. Heat-treated cells and heat-stable secreted molecules trigger multiple strain-specific activities explaining the therapeutic efficacy of L. acidophilus LB. This review discusses the current body of knowledge on the antimicrobial mechanisms of action exerted by L. acidophilus LB demonstrated in in vitro and in vivo experimental studies, and the evidence for the therapeutic efficacy of this anti-infectious biotherapeutic agent proved in randomized clinical trials for the treatment of acute and persistent watery diarrhoea associated with several intestinal infectious diseases in humans.

  20. Immunogenicity to Biotherapeutics – the role of Anti-drug Immune complexes

    Directory of Open Access Journals (Sweden)

    Murli eKrishna

    2016-02-01

    Full Text Available AbstractBiologic molecules are increasingly becoming a part of the therapeutics portfolio that has been either recently approved for marketing or those that are in the pipeline of several biotech and pharmaceutical companies. This is largely based on their ability to be highly specific relative to small molecules. However by virtue of being a large protein, and having a complex structure with structural variability arising from production using recombinant gene technology in cell lines, such therapeutics run the risk of being recognized as foreign by a host immune system. Given the range of immune mediated adverse effects that have been documented to biologic drugs thus far, including infusion reactions, and the evolving therapeutic platforms in the pipeline that engineer different functional modules in a biotherapeutic, it is critical to understand the interplay of the adaptive and innate immune responses, the pathophysiology of immunogenicity to biologic drugs in instances where there have been immune mediated adverse clinical sequelae and address technical approaches for their laboratory evaluation. The current paradigm in immunogenicity evaluation has a tiered approach to the detection and characterization of anti-drug antibodies (ADAs elicited in vivo to a biotherapeutic; alongside with the structural, biophysical and molecular information of the therapeutic, these analytical assessments form the core of the immunogenicity risk assessment. However many of the immune mediated adverse effects attributed to ADAs require the formation of a drug/ADA immune complex intermediate (ICs that can have a variety of downstream effects. This review will focus on the activation of potential immunopathological pathways arising as a consequence of circulating as well as cell surface bound drug bearing-ICs, risk factors that are either intrinsic to the therapeutic molecule or to the host which might predispose to IC mediated effects, and review the recent

  1. Immunogenicity to Biotherapeutics – The Role of Anti-drug Immune Complexes

    Science.gov (United States)

    Krishna, Murli; Nadler, Steven G.

    2016-01-01

    Biological molecules are increasingly becoming a part of the therapeutics portfolio that has been either recently approved for marketing or those that are in the pipeline of several biotech and pharmaceutical companies. This is largely based on their ability to be highly specific relative to small molecules. However, by virtue of being a large protein, and having a complex structure with structural variability arising from production using recombinant gene technology in cell lines, such therapeutics run the risk of being recognized as foreign by a host immune system. In the context of immune-mediated adverse effects that have been documented to biological drugs thus far, including infusion reactions, and the evolving therapeutic platforms in the pipeline that engineer different functional modules in a biotherapeutic, it is critical to understand the interplay of the adaptive and innate immune responses, the pathophysiology of immunogenicity to biological drugs in instances where there have been immune-mediated adverse clinical sequelae and address technical approaches for their laboratory evaluation. The current paradigm in immunogenicity evaluation has a tiered approach to the detection and characterization of anti-drug antibodies (ADAs) elicited in vivo to a biotherapeutic; alongside with the structural, biophysical, and molecular information of the therapeutic, these analytical assessments form the core of the immunogenicity risk assessment. However, many of the immune-mediated adverse effects attributed to ADAs require the formation of a drug/ADA immune complex (IC) intermediate that can have a variety of downstream effects. This review will focus on the activation of potential immunopathological pathways arising as a consequence of circulating as well as cell surface bound drug bearing ICs, risk factors that are intrinsic either to the therapeutic molecule or to the host that might predispose to IC-mediated effects, and review the recent literature on

  2. Biotransformation and in vivo stability of protein biotherapeutics: impact on candidate selection and pharmacokinetic profiling.

    Science.gov (United States)

    Hall, Michael P

    2014-11-01

    Historically, since the metabolism of administered peptide/protein drugs ("biotherapeutics") has been expected to undergo predictable pathways similar to endogenous proteins, comprehensive biotherapeutic metabolism studies have not been widely reported in the literature. However, since biotherapeutics have rapidly evolved into an impressive array of eclectic modalities, there has been a shift toward understanding the impact of metabolism on biotherapeutic development. For biotherapeutics containing non-native chemical linkers and other moieties besides natural amino acids, metabolism studies are critical as these moieties may impart undesired toxicology. For biotherapeutics that are composed solely of natural amino acids, where end-stage peptide and amino acid catabolites do not generally pose toxicity concerns, the understanding of biotherapeutic biotransformation, defined as in vivo modifications such as peripherally generated intermediate circulating catabolites prior to end-stage degradation or elimination, may impact in vivo stability and potency/clearance. As of yet, there are no harmonized methodologies for understanding biotherapeutic biotransformation and its impact on drug development, nor is there clear guidance from regulatory agencies on how and when these studies should be conducted. This review provides an update on biotherapeutic biotransformation studies and an overview of lessons learned, tools that have been developed, and suggestions of approaches to address issues. Biotherapeutic biotransformation studies, especially for certain modalities, should be implemented at an early stage of development to 1) understand the impact on potency/clearance, 2) select the most stable candidates or direct protein re-engineering efforts, and 3) select the best bioanalytical technique(s) for proper drug quantification and subsequent pharmacokinetic profiling and exposure/response assessment. Copyright © 2014 by The American Society for Pharmacology and

  3. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    Science.gov (United States)

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination.

  4. Light scattering application for bacterial cell monitoring during cultivation process

    Science.gov (United States)

    Kotsyumbas, Igor Ya.; Kushnir, Igor M.; Bilyy, Rostyslav O.; Yarynovska, Ivanna H.; Getman, Vasyl'B.; Bilyi, Alexander I.

    2007-07-01

    Monitoring of bacterial cell numbers is of great importance not only in microbiological industry but also for control of liquids contamination in the food and pharmaceutical industries. Here we describe a novel low-cost and highly efficient technology for bacterial cell monitoring during cultivation process. The technology incorporates previously developed monitoring device and algorithm of its action. The devise analyses light scattered by suspended bacterial cells. Current stage utilizes monochromatic coherent light and detects amplitudes and durations of scattered light impulses, it does not require any labeling of bacterial cell. The system is calibrated using highly purificated bacteria-free water as standard. Liquid medial are diluted and analyzed by the proposed technology to determine presence of bacteria. Detection is done for a range of particle size from 0.1 to 10 μm, and thus particles size distribution is determined. We analyzed a set of different bacterial suspensions and also their changes in quantity and size distribution during cultivation. Based on the obtained results we conclude that proposed technology can be very effective for bacteria monitoring during cultivation process, providing benefits of low simplicity and low cost of analysis with simultaneous high detection precision.

  5. Micro-magnet arrays for specific single bacterial cell positioning

    Energy Technology Data Exchange (ETDEWEB)

    Pivetal, Jérémy, E-mail: jeremy.piv@netcmail.com [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France); Royet, David [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France); Ciuta, Georgeta [Univ. Grenoble Alpes, Inst NEEL, F-38042 Grenoble (France); CNRS, Inst NEEL, F-38042 Grenoble (France); Frenea-Robin, Marie [Université de Lyon, Université Lyon 1, CNRS UMR 5005, Laboratoire Ampère, F-69622 Villeurbanne (France); Haddour, Naoufel [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France); Dempsey, Nora M. [Univ. Grenoble Alpes, Inst NEEL, F-38042 Grenoble (France); CNRS, Inst NEEL, F-38042 Grenoble (France); Dumas-Bouchiat, Frédéric [Univ Limoges, CNRS, SPCTS UMR 7513, 12 Rue Atlantis, F-87068 Limoges (France); Simonet, Pascal [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France)

    2015-04-15

    In various contexts such as pathogen detection or analysis of microbial diversity where cellular heterogeneity must be taken into account, there is a growing need for tools and methods that enable microbiologists to analyze bacterial cells individually. One of the main challenges in the development of new platforms for single cell studies is to perform precise cell positioning, but the ability to specifically target cells is also important in many applications. In this work, we report the development of new strategies to selectively trap single bacterial cells upon large arrays, based on the use of micro-magnets. Escherichia coli bacteria were used to demonstrate magnetically driven bacterial cell organization. In order to provide a flexible approach adaptable to several applications in the field of microbiology, cells were magnetically and specifically labeled using two different strategies, namely immunomagnetic labeling and magnetic in situ hybridization. Results show that centimeter-sized arrays of targeted, isolated bacteria can be successfully created upon the surface of a flat magnetically patterned hard magnetic film. Efforts are now being directed towards the integration of a detection tool to provide a complete micro-system device for a variety of microbiological applications. - Highlights: 1.We report a new approach to selectively micropattern bacterial cells individually upon micro-magnet arrays. 2.Permanent micro-magnets of a size approaching that of bacteria could be fabricated using a Thermo-Magnetic Patterning process. 3.Bacterial cells were labeled using two different magnetic labeling strategies providing flexible approach adaptable to several applications in the field of microbiology.

  6. Measuring biotherapeutic viscosity and degradation on-chip with particle diffusometry.

    Science.gov (United States)

    Clayton, K N; Lee, D; Wereley, S T; Kinzer-Ursem, T L

    2017-11-21

    In the absence of efficient ways to test drug stability and efficacy, pharmaceuticals that have been stored outside of set temperature conditions are destroyed, often at great cost. This is especially problematic for biotherapeutics, which are highly sensitive to temperature fluctuations. Current platforms for assessing the stability of protein-based biotherapeutics in high throughput and in low volumes are unavailable outside of research and development laboratories and are not efficient for use in production, quality control, distribution, or clinical settings. In these alternative environments, microanalysis platforms could provide significant advantages for the characterization of biotherapeutic degradation. Here we present particle diffusometry (PD), a new technique to study degradation of biotherapeutic solutions. PD uses a simple microfluidic chip and microscope setup to calculate the Brownian motion of particles in a quiescent solution using a variation of particle image velocimetry (PIV) fundamentals. We show that PD can be used to measure the viscosity of protein solutions to discriminate native protein from degraded samples as well as to determine the change in viscosity as a function of therapeutic concentration. PD viscosity analysis is applied to two particularly important biotherapeutic preparations: insulin, a commonly used protein for diabetic patients, and monoclonal antibodies which are an emerging class of biotherapeutics used to treat a variety of diseases such as autoimmune disorders and cancer. PD-based characterization of solution viscosity is a new tool for biotherapeutic analysis, and owing to its easy setup could readily be implemented at key points of the pharmaceutical delivery chain and in clinical settings.

  7. Softness of the bacterial cell wall of Streptococcus mitis as probed by microelectrophoresis

    NARCIS (Netherlands)

    Rodriguez, VV; Busscher, HJ; Norde, W; van der Mei, HC

    Chemical and structural complexity of bacterial cell surfaces complicate accurate quantification of cell surfaces properties. The presence of fibrils, fimbriae or other surface appendages on bacterial cell surfaces largely influence those properties and would therefore play a major function in

  8. Softness of the bacterial cell wall of Streptococcus mitis as probed by micro-electrophoresis

    NARCIS (Netherlands)

    Vadillo-Rodriguez, V.; Busscher, H.J.; Norde, W.; Mei, van der H.C.

    2002-01-01

    Chemical and structural complexity of bacterial cell surfaces complicate accurate quantification of cell surfaces properties. The presence of fibrils, fimbriae or other surface appendages on bacterial cell surfaces largely influence those properties and would therefore play a major function in

  9. Elucidating Duramycin’s Bacterial Selectivity and Mode of Action on the Bacterial Cell Envelope

    Directory of Open Access Journals (Sweden)

    Sahar Hasim

    2018-02-01

    Full Text Available The use of naturally occurring antimicrobial peptides provides a promising route to selectively target pathogenic agents and to shape microbiome structure. Lantibiotics, such as duramycin, are one class of bacterially produced peptidic natural products that can selectively inhibit the growth of other bacteria. However, despite longstanding characterization efforts, the microbial selectivity and mode of action of duramycin are still obscure. We describe here a suite of biological, chemical, and physical characterizations that shed new light on the selective and mechanistic aspects of duramycin activity. Bacterial screening assays have been performed using duramycin and Populus-derived bacterial isolates to determine species selectivity. Lipidomic profiles of selected resistant and sensitive strains show that the sensitivity of Gram-positive bacteria depends on the presence of phosphatidylethanolamine (PE in the cell membrane. Further the surface and interface morphology were studied by high resolution atomic force microscopy and showed a progression of cellular changes in the cell envelope after treatment with duramycin for the susceptible bacterial strains. Together, these molecular and cellular level analyses provide insight into duramycin’s mode of action and a better understanding of its selectivity.

  10. Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells.

    Science.gov (United States)

    Martín, César; Etxaniz, Asier; Uribe, Kepa B; Etxebarria, Aitor; González-Bullón, David; Arlucea, Jon; Goñi, Félix M; Aréchaga, Juan; Ostolaza, Helena

    2015-09-08

    Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of "toxin-coated bacteria" proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or "free" in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca(2+)-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system.

  11. Isolation of cell-free bacterial inclusion bodies.

    Science.gov (United States)

    Rodríguez-Carmona, Escarlata; Cano-Garrido, Olivia; Seras-Franzoso, Joaquin; Villaverde, Antonio; García-Fruitós, Elena

    2010-09-17

    Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for inclusion body isolation from Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of inclusion bodies in biological interfaces. Using an appropriate combination of chemical and mechanical cell disruption methods we have established a convenient procedure for the recovery of bacterial inclusion bodies with undetectable levels of viable cell contamination, below 10⁻¹ cfu/ml, keeping the particulate organization of these aggregates regarding size and protein folding features. The application of the developed protocol allows obtaining bacterial free inclusion bodies suitable for use in mammalian cell cultures and other biological interfaces.

  12. Electrostatic behavior of the charge-regulated bacterial cell surface.

    Science.gov (United States)

    Hong, Yongsuk; Brown, Derick G

    2008-05-06

    The electrostatic behavior of the charge-regulated surfaces of Gram-negative Escherichia coli and Gram-positive Bacillus brevis was studied using numerical modeling in conjunction with potentiometric titration and electrophoretic mobility data as a function of solution pH and electrolyte composition. Assuming a polyelectrolytic polymeric bacterial cell surface, these experimental and numerical analyses were used to determine the effective site numbers of cell surface acid-base functional groups and Ca(2+) sorption coefficients. Using effective site concentrations determined from 1:1 electrolyte (NaCl) experimental data, the charge-regulation model was able to replicate the effects of 2:1 electrolyte (CaCl(2)), both alone and as a mixture with NaCl, on the measured zeta potential using a single Ca(2+) surface binding constant for each of the bacterial species. This knowledge is vital for understanding how cells respond to changes in solution pH and electrolyte composition as well as how they interact with other surfaces. The latter is especially important due to the widespread use of the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory in the interpretation of bacterial adhesion. As surface charge and surface potential both vary on a charge-regulated surface, accurate modeling of bacterial interactions with surfaces ultimately requires use of an electrostatic model that accounts for the charge-regulated nature of the cell surface.

  13. Modeling base excision repair in Escherichia coli bacterial cells

    International Nuclear Information System (INIS)

    Belov, O.V.

    2011-01-01

    A model describing the key processes in Escherichia coli bacterial cells during base excision repair is developed. The mechanism is modeled of damaged base elimination involving formamidopyrimidine DNA glycosylase (the Fpg protein), which possesses several types of activities. The modeling of the transitions between DNA states is based on a stochastic approach to the chemical reaction description

  14. Development of bacterial cell-based system for intracellular ...

    African Journals Online (AJOL)

    Development of bacterial cell-based system for intracellular antioxidant activity screening assay using green fluorescence protein (GFP) reporter. ... Both strains demonstrated that quercetin and α- tocopherol exhibited the most potent and significant antioxidant activity with more than 60% reduction of intracellular superoxide ...

  15. Effect of radiation on bacterial cells pathogenesis

    International Nuclear Information System (INIS)

    Szulc, M.; Tropilo, J.; Zajaczkowska, E.

    1983-01-01

    In order to determine the effect of X rays on the pathogeny of Erysipelothrix rhusiopathiae and Pasteurella multocida 334 mice were infected subcutaneously with the germs exposed to 10 Gy and D 10 . It was found that a single exposition of E. rhusiopathiae and P. multocida to 10 Gy and D 10 did not change pathogenic properties of these cells. P. multocida showed higher sensitivity to X rays: D 10 for that species was 94 Gy and for E. rhusiopathiae - 156 Gy. (author)

  16. BACTERIAL CELL KILLING MEDIATED BY TOPOISOMERASE I DNA CLEAVAGE ACTIVITY

    Science.gov (United States)

    Cheng, Bokun; Shukla, Shikha; Vasunilashorn, Sarinnapha; Mukhopadhyay, Somshuvra; Tse-Dinh, Yuk-Ching

    2005-01-01

    DNA topoisomerases are important clinical targets for antibacterial and anticancer therapy. At least one type IA DNA topoisomerases can be found in every bacterium, making it a logical target for antibacterial agents that can convert the enzyme into poison by trapping its covalent complex with DNA. However, it has not been possible previously to observe the consequence of having such stabilized covalent complex of bacterial topoisomerase I in vivo. We isolated a mutant of recombinant Yersinia pestis topoisomerase I that forms a stabilized covalent complex with DNA by screening for the ability to induce the SOS response in Escherichia coli. Overexpression of this mutant topoisomerase I resulted in bacterial cell death. From sequence analysis and site-directed mutagenesis, it was determined that a single amino acid substitution in the TOPRIM domain changing a strictly conserved glycine residue to serine in either the Y. pestis or E. coli topoisomerase I can result in a mutant enzyme that has the SOS inducing and cell killing properties. Analysis of the purified mutant enzymes showed that they have no relaxation activity but retain the ability to cleave DNA and form a covalent complex. These results demonstrate that perturbation of the active site region of bacterial topoisomerase I can result in stabilization of the covalent intermediate, with the in vivo consequence of bacterial cell death. Small molecules that induce similar perturbation in the enzyme-DNA complex should be candidates as leads for novel antibacterial agents. PMID:16159875

  17. 75 FR 63188 - Draft Guidance for Industry: Early Clinical Trials With Live Biotherapeutic Products: Chemistry...

    Science.gov (United States)

    2010-10-14

    ...] Draft Guidance for Industry: Early Clinical Trials With Live Biotherapeutic Products: Chemistry...: Chemistry, Manufacturing, and Control Information'' dated September 2010. The draft guidance provides... Products: Chemistry, Manufacturing, and Control Information'' dated September 2010. The draft guidance...

  18. Optimizing the Bioavailability of Subcutaneously Administered Biotherapeutics Through Mechanochemical Drivers.

    Science.gov (United States)

    Collins, D S; Kourtis, L C; Thyagarajapuram, N R; Sirkar, R; Kapur, S; Harrison, M W; Bryan, D J; Jones, G B; Wright, J M

    2017-10-01

    The subcutaneous route offers myriad benefits for the administration of biotherapeutics in both acute and chronic diseases, including convenience, cost effectiveness and the potential for automation through closed-loop systems. Recent advances in parenteral administration devices and the use of additives which enhance drug dispersion have generated substantial additional interest in IV to SQ switching studies. Designing pre-clinical and clinical studies using SQ mediated delivery however requires deep understanding of complex inter-related physiologies and transport pathways governing the interstitial matrix, vascular system and lymphatic channels. This expert review will highlight key structural features which contribute to transport and biodistribution in the subcutaneous space and also assess the impact of drug formulations. Based on the rapidly growing interest in the SQ delivery route, a number of potential areas for future development are highlighted, which are likely to allow continued evolution and innovation in this important area.

  19. Reducing or Eliminating Polysorbate Induced Anaphylaxis and Unwanted Immunogenicity in Biotherapeutics

    Directory of Open Access Journals (Sweden)

    Edward Maggio

    2017-09-01

    Full Text Available The increasing use of biotherapeutics across a growing spectrum of neoplastic, autoimmune, and inflammatory diseases has resulted in a corresponding increase in hypersensitivity reactions. The origins of anaphylaxis are often attributed to undefined intrinsic properties of the biotherapeutic protein itself, ignoring the broader potential negative contributions of functional excipients, in particular polyoxyethylene containing surfactants such as polysorbate 80 and polysorbate 20 (Tween 80 and Tween 20. These surfactants allow biotherapeutics to meet the stringent challenges of extended shelf-life, increased solubility, protein aggregation prevention, reduced administration volume, and satisfactory reconstitution properties in the case of lyophilized biotherapeutics. The potential negative impact of certain functional excipients on product performance characteristics such as anaphylaxis and immunogenicity is often overlooked. While regulatory authorities understandably focus heavily on comparable efficacy in evaluating biosimilars, similar efficacy does not necessarily imply a similar safety profile between the originator and biosimilar products. Both unwanted immunogenicity and anaphylaxis do comprise major components of safety assessment, however, few if any attempts are made to differentiate drug-related from excipient-related anaphylaxis. Replacement of anaphylactogenic and immunogenic functional excipients with equally effective but safer alternatives will allow biotherapeutic developers to differentiate their biotherapeutic, biosimilar, or biobetter from the large number of nearly identical competitor products, simultaneously providing a substantial commercial benefit as well as critical clinical benefits for all concerned – patients, physicians, and third party payers.

  20. WHO standards for biotherapeutics, including biosimilars: an example of the evaluation of complex biological products.

    Science.gov (United States)

    Knezevic, Ivana; Griffiths, Elwyn

    2017-11-01

    The most advanced regulatory processes for complex biological products have been put in place in many countries to provide appropriate regulatory oversight of biotherapeutic products in general, and similar biotherapeutics in particular. This process is still ongoing and requires regular updates to national regulatory requirements in line with scientific developments and up-to-date standards. For this purpose, strong knowledge of and expertise in evaluating biotherapeutics in general and similar biotherapeutic products, also called biosimilars, in particular is essential. Here, we discuss the World Health Organization's international standard-setting role in the regulatory evaluation of recombinant DNA-derived biotherapeutic products, including biosimilars, and provide examples that may serve as models for moving forward with nonbiological complex medicinal products. A number of scientific challenges and regulatory considerations imposed by the advent of biosimilars are described, together with the lessons learned, to stimulate future discussions on this topic. In addition, the experiences of facilitating the implementation of guiding principles for evaluation of similar biotherapeutic products into regulatory and manufacturers' practices in various countries over the past 10 years are briefly explained, with the aim of promoting further developments and regulatory convergence of complex biological and nonbiological products. © 2017 The Authors. Annals of the New York Academy of Sciences. The World Health Organization retains copyright and all other rights in the manuscript of this article as submitted for publication.

  1. Large-scale production of pharmaceutical proteins in plant cell culture-the Protalix experience.

    Science.gov (United States)

    Tekoah, Yoram; Shulman, Avidor; Kizhner, Tali; Ruderfer, Ilya; Fux, Liat; Nataf, Yakir; Bartfeld, Daniel; Ariel, Tami; Gingis-Velitski, Svetlana; Hanania, Uri; Shaaltiel, Yoseph

    2015-10-01

    Protalix Biotherapeutics develops recombinant human proteins and produces them in plant cell culture. Taliglucerase alfa has been the first biotherapeutic expressed in plant cells to be approved by regulatory authorities around the world. Other therapeutic proteins are being developed and are currently at various stages of the pipeline. This review summarizes the major milestones reached by Protalix Biotherapeutics to enable the development of these biotherapeutics, including platform establishment, cell line selection, manufacturing process and good manufacturing practice principles to consider for the process. Examples of the various products currently being developed are also presented. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  2. Codon Optimization in the Production of Recombinant Biotherapeutics: Potential Risks and Considerations.

    Science.gov (United States)

    Mauro, Vincent P

    2018-02-01

    Biotherapeutics are increasingly becoming the mainstay in the treatment of a variety of human conditions, particularly in oncology and hematology. The production of therapeutic antibodies, cytokines, and fusion proteins have markedly accelerated these fields over the past decade and are probably the major contributor to improved patient outcomes. Today, most protein therapeutics are expressed as recombinant proteins in mammalian cell lines. An expression technology commonly used to increase protein levels involves codon optimization. This approach is possible because degeneracy of the genetic code enables most amino acids to be encoded by more than one synonymous codon and because codon usage can have a pronounced influence on levels of protein expression. Indeed, codon optimization has been reported to increase protein expression by >  1000-fold. The primary tactic of codon optimization is to increase the rate of translation elongation by overcoming limitations associated with species-specific differences in codon usage and transfer RNA (tRNA) abundance. However, in mammalian cells, assumptions underlying codon optimization appear to be poorly supported or unfounded. Moreover, because not all synonymous codon mutations are neutral, codon optimization can lead to alterations in protein conformation and function. This review discusses codon optimization for therapeutic protein production in mammalian cells.

  3. Vaginal epithelial cells regulate membrane adhesiveness to co-ordinate bacterial adhesion

    NARCIS (Netherlands)

    Younes, Jessica A.; Klappe, Karin; Kok, Jan Willem; Busscher, Henk J.; Reid, Gregor; van der Mei, Henny C.

    Vaginal epithelium is colonized by different bacterial strains and species. The bacterial composition of vaginal biofilms controls the balance between health and disease. Little is known about the relative contribution of the epithelial and bacterial cell surfaces to bacterial adhesion and whether

  4. Stoichiometry of mercury-thiol complexes on bacterial cell envelopes

    Energy Technology Data Exchange (ETDEWEB)

    Mishra, Bhoopesh; Shoenfelt, Elizabeth; Yu, Qiang; Yee, Nathan; Fein, Jeremy B.; Myneni, Satish C. B.

    2017-08-01

    We have examined the speciation of Hg(II) complexed with intact cell suspensions (1013 cells L- 1) of Bacillus subtilis, a common gram-positive soil bacterium, Shewanella oneidensis MR-1, a facultative gram-negative aquatic organism, and Geobacter sulfurreducens, a gram-negative anaerobic bacterium capable of Hg-methylation at Hg(II) loadings spanning four orders of magnitude (120 nM to 350 μM) at pH 5.5 (± 0.2). The coordination environments of Hg on bacterial cells were analyzed using synchrotron based X-ray Absorption Near Edge Structure (XANES) and Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy at the Hg LIII edge. The abundance of thiols on intact cells was determined by a fluorescence-spectroscopy based method using a soluble bromobimane, monobromo(trimethylammonio)bimane (qBBr) to block thiol sites, and potentiometric titrations of biomass with and without qBBr treatment. The chemical forms of S on intact bacterial cells were determined using S k-edge XANES spectroscopy.

  5. Virus and Bacterial Cell Chemical Analysis by NanoSIMS

    Energy Technology Data Exchange (ETDEWEB)

    Weber, P; Holt, J

    2008-07-28

    In past work for the Department of Homeland Security, the LLNL NanoSIMS team has succeeded in extracting quantitative elemental composition at sub-micron resolution from bacterial spores using nanometer-scale secondary ion mass spectrometry (NanoSIMS). The purpose of this task is to test our NanoSIMS capabilities on viruses and bacterial cells. This initial work has proven successful. We imaged Tobacco Mosaic Virus (TMV) and Bacillus anthracis Sterne cells using scanning electron microscopy (SEM) and then analyzed those samples by NanoSIMS. We were able resolve individual viral particles ({approx}18 nm by 300 nm) in the SEM and extract correlated elemental composition in the NanoSIMS. The phosphorous/carbon ratio observed in TMV is comparable to that seen in bacterial spores (0.033), as was the chlorine/carbon ratio (0.11). TMV elemental composition is consistent from spot to spot, and TMV is readily distinguished from debris by NanoSIMS analysis. Bacterial cells were readily identified in the SEM and relocated in the NanoSIMS for elemental analysis. The Ba Sterne cells were observed to have a measurably lower phosphorous/carbon ratio (0.005), as compared to the spores produced in the same run (0.02). The chlorine/carbon ratio was approximately 2.5X larger in the cells (0.2) versus the spores (0.08), while the fluorine/carbon ratio was approximately 10X lower in the cells (0.008) than the spores (0.08). Silicon/carbon ratios for both cells and spores encompassed a comparable range. The initial data in this study suggest that high resolution analysis is useful because it allows the target agent to be analyzed separate from particulates and other debris. High resolution analysis would also be useful for trace sample analysis. The next step in this work is to determine the potential utility of elemental signatures in these kinds of samples. We recommend bulk analyses of media and agent samples to determine the range of media compositions in use, and to determine how

  6. Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

    OpenAIRE

    Romaniuk, Joseph A. H.; Cegelski, Lynette

    2015-01-01

    The ability to characterize bacterial cell-wall composition and structure is crucial to understanding the function of the bacterial cell wall, determining drug modes of action and developing new-generation therapeutics. Solid-state NMR has emerged as a powerful tool to quantify chemical composition and to map cell-wall architecture in bacteria and plants, even in the context of unperturbed intact whole cells. In this review, we discuss solid-state NMR approaches to define pe...

  7. Tiny cells meet big questions: a closer look at bacterial cell biology.

    Science.gov (United States)

    Goley, Erin D

    2013-04-01

    While studying actin assembly as a graduate student with Matt Welch at the University of California at Berkeley, my interest was piqued by reports of surprising observations in bacteria: the identification of numerous cytoskeletal proteins, actin homologues fulfilling spindle-like functions, and even the presence of membrane-bound organelles. Curiosity about these phenomena drew me to Lucy Shapiro's lab at Stanford University for my postdoctoral research. In the Shapiro lab, and now in my lab at Johns Hopkins, I have focused on investigating the mechanisms of bacterial cytokinesis. Spending time as both a eukaryotic cell biologist and a bacterial cell biologist has convinced me that bacterial cells present the same questions as eukaryotic cells: How are chromosomes organized and accurately segregated? How is force generated for cytokinesis? How is polarity established? How are signals transduced within and between cells? These problems are conceptually similar between eukaryotes and bacteria, although their solutions can differ significantly in specifics. In this Perspective, I provide a broad view of cell biological phenomena in bacteria, the technical challenges facing those of us who peer into bacterial cells, and areas of common ground as research in eukaryotic and bacterial cell biology moves forward.

  8. Microfabricated ratchet structures for concentrating and patterning motile bacterial cells

    International Nuclear Information System (INIS)

    Kim, Sang Yub; Lee, Eun Se; Lee, Ho Jae; Lee, Se Yeon; Lee, Sung Kuk; Kim, Taesung

    2010-01-01

    We present a novel microfabricated concentrator for Escherichia coli that can be a stand-alone and self-contained microfluidic device because it utilizes the motility of cells. First of all, we characterize the motility of E. coli cells and various ratcheting structures that can guide cells to move in a desired direction in straight and circular channels. Then, we combine these ratcheting microstructures with the intrinsic tendency of cells to swim on the right side in microchannels to enhance the concentration rates up to 180 fold until the concentrators are fully filled with cells. Furthermore, we demonstrate that cells can be positioned and concentrated with a constant spacing distance on a surface, allowing spatial patterning of motile cells. These results can be applied to biosorption or biosensor devices that are powered by motile cells because they can be highly concentrated without any external mechanical and electrical energy sources. Hence, we believe that the concentrator design holds considerable potential to be applied for concentrating and patterning other motile microbes and providing a versatile structure for motility study of bacterial cells.

  9. Bending forces plastically deform growing bacterial cell walls

    Science.gov (United States)

    Amir, Ariel; Babaeipour, Farinaz; McIntosh, Dustin B.; Nelson, David R.; Jun, Suckjoon

    2014-01-01

    Cell walls define a cell’s shape in bacteria. The walls are rigid to resist large internal pressures, but remarkably plastic to adapt to a wide range of external forces and geometric constraints. Currently, it is unknown how bacteria maintain their shape. In this paper, we develop experimental and theoretical approaches and show that mechanical stresses regulate bacterial cell wall growth. By applying a precisely controllable hydrodynamic force to growing rod-shaped Escherichia coli and Bacillus subtilis cells, we demonstrate that the cells can exhibit two fundamentally different modes of deformation. The cells behave like elastic rods when subjected to transient forces, but deform plastically when significant cell wall synthesis occurs while the force is applied. The deformed cells always recover their shape. The experimental results are in quantitative agreement with the predictions of the theory of dislocation-mediated growth. In particular, we find that a single dimensionless parameter, which depends on a combination of independently measured physical properties of the cell, can describe the cell’s responses under various experimental conditions. These findings provide insight into how living cells robustly maintain their shape under varying physical environments. PMID:24711421

  10. Factors affecting daughter cells' arrangement during the early bacterial divisions.

    Directory of Open Access Journals (Sweden)

    Pin-Tzu Su

    Full Text Available On agar plates, daughter cells of Escherichia coli mutually slide and align side-by-side in parallel during the first round of binary fission. This phenomenon has been previously attributed to an elastic material that restricts apparently separated bacteria from being in string. We hypothesize that the interaction between bacteria and the underneath substratum may affect the arrangement of the daughter bacteria. To test this hypothesis, bacterial division on hyaluronic acid (HA gel, as an alternative substratum, was examined. Consistent with our proposition, the HA gel differs from agar by suppressing the typical side-by-side alignments to a rare population. Examination of bacterial surface molecules that may contribute to the daughter cells' arrangement yielded an observation that, with disrupted lpp, the E. coli daughter cells increasingly formed non-typical patterns, i.e. neither sliding side-by-side in parallel nor forming elongated strings. Therefore, our results suggest strongly that the early cell patterning is affected by multiple interaction factors. With oscillatory optical tweezers, we further demonstrated that the interaction force decreased in bacteria without Lpp, a result substantiating our notion that the side-by-side sliding phenomenon directly reflects the strength of in-situ interaction between bacteria and substratum.

  11. Metabolic activity of bacterial cell enumerated by direct viable count

    International Nuclear Information System (INIS)

    Roszak, D.B.; Colwell, R.R.

    1987-01-01

    The direct viable count (DVC) method was modified by incorporation radiolabeled substrates in microautoradiographic analyses to assess bacterial survival in controlled laboratory microcosms. The DVC method, which permits enumeration of culturable and nonculturable cells, discriminates those cells that are responsive to added nutrients but in which division is inhibited by the addition of nalidixic acid. The resulting elongated cells represent all viable cells; this includes those that are culturable on routine media and those that are not. Escherichia coli and Salmonella enteritidis were employed in the microcosm studies, and radiolabeled substrates included [methyl- 3 H] thymidine or [U- 14 C] glutamic acid. Samples taken at selected intervals during the survival experiments were examined by epifluorescence microscopy to enumerate cells by the DVC and acridine orange direct count methods, as well as by culture methods. Good correlation was obtained for cell-associated metabolic activity, measured by microautoradiography and substrate responsiveness (by the DVC method) at various stages of survival. Of the cells responsive to nutrients by the DVC method, ca. 90% were metabolically active by the microautoradiographic method. No significant difference was observed between DVC enumerations with or without added radiolabeled substrate

  12. Vaginal epithelial cells regulate membrane adhesiveness to co-ordinate bacterial adhesion.

    Science.gov (United States)

    Younes, Jessica A; Klappe, Karin; Kok, Jan Willem; Busscher, Henk J; Reid, Gregor; van der Mei, Henny C

    2016-04-01

    Vaginal epithelium is colonized by different bacterial strains and species. The bacterial composition of vaginal biofilms controls the balance between health and disease. Little is known about the relative contribution of the epithelial and bacterial cell surfaces to bacterial adhesion and whether and how adhesion is regulated over cell membrane regions. Here, we show that bacterial adhesion forces with cell membrane regions not located above the nucleus are stronger than with regions above the nucleus both for vaginal pathogens and different commensal and probiotic lactobacillus strains involved in health. Importantly, adhesion force ratios over membrane regions away from and above the nucleus coincided with the ratios between numbers of adhering bacteria over both regions. Bacterial adhesion forces were dramatically decreased by depleting the epithelial cell membrane of cholesterol or sub-membrane cortical actin. Thus, epithelial cells can regulate membrane regions to which bacterial adhesion is discouraged, possibly to protect the nucleus. © 2015 John Wiley & Sons Ltd.

  13. Free and total biotherapeutic evaluation in chromatographic assays: interference from targets and immunogenicity.

    Science.gov (United States)

    White, Joleen T; Bonilla, Leo E

    2012-10-01

    Measurement of drug concentrations is critical during drug development, supporting evaluation of safety and efficacy in the context of pharmacokinetics. Protein-based therapeutics have been historically measured by immunoassay methods. Technological advances provide new opportunities to measure these biotherapeutics using previously incompatible chromatographic techniques, such as MS. These advances are breaking down the barriers between 'large-molecule' and 'small-molecule' bioanalysis, and pushing scientists outside their comfort zones. One challenge in measuring biotherapeutic concentration is potential impact from other matrix components, such as therapeutic target or antidrug antibodies. Depending on the specific assay development objective, target interference could be either desired (favoring free measurement) or undesired (favoring total measurement). Orthogonal techniques provide additional tools to meet this challenge. The goal of this review is to introduce both small- and large-molecule bioanalytical scientists to the opportunities and challenges to consider while evaluating orthogonal methods for biotherapeutic bioanalysis.

  14. Lung Dendritic Cells Facilitate Extrapulmonary Bacterial Dissemination during Pneumococcal Pneumonia

    Directory of Open Access Journals (Sweden)

    Alva eRosendahl

    2013-06-01

    Full Text Available Streptococcus pneumoniae is a leading cause of bacterial pneumonia worldwide. Given the critical role of dendritic cells (DCs in regulating and modulating the immune response to pathogens, we investigated here the role of DCs in S. pneumoniae lung infections. Using a well-established transgenic mouse line which allows the conditional transient depletion of DCs, we showed that ablation of DCs resulted in enhanced resistance to intranasal challenge with S. pneumoniae. DC-depleted mice exhibited delayed bacterial systemic dissemination, significantly reduced bacterial loads in the infected organs and lower levels of serum inflammatory mediators than non-depleted animals. The increased resistance of DC-depleted mice to S. pneumoniae was associated with a better capacity to restrict pneumococci extrapulmonary dissemination. Furthermore, we demonstrated that S. pneumoniae disseminated from the lungs into the regional lymph nodes in a cell-independent manner and that this direct way of dissemination was much more efficient in the presence of DCs. We also provide evidence that S. pneumoniae induces expression and activation of matrix metalloproteinase-9 (MMP-9 in cultured bone marrow-derived DCs. MMP-9 is a protease involved in the breakdown of extracellular matrix proteins and is critical for DC trafficking across extracellular matrix and basement membranes during the migration from the periphery to the lymph nodes. MMP-9 was also significantly up-regulated in the lungs of mice after intranasal infection with S. pneumoniae. Notably, the expression levels of MMP-9 in the infected lungs were significantly decreased after depletion of DCs suggesting the involvement of DCs in MMP-9 production during pneumococcal pneumonia. Thus, we propose that S. pneumoniae can exploit the DC-derived proteolysis to open tissue barriers thereby facilitating its own dissemination from the local site of infection.

  15. Segrosome complex formation during DNA trafficking in bacterial cell division

    Directory of Open Access Journals (Sweden)

    Maria A. Oliva

    2016-09-01

    Full Text Available Bacterial extrachromosomal DNAs often contribute to virulence in pathogenic organisms or facilitate adaptation to particular environments. The transmission of genetic information from one generation to the next requires sufficient partitioning of DNA molecules to ensure that at least one copy reaches each side of the division plane and is inherited by the daughter cells. Segregation of the bacterial chromosome occurs during or after replication and probably involves a strategy in which several protein complexes participate to modify the folding pattern and distribution first of the origin domain and then of the rest of the chromosome. Low-copy number plasmids rely on specialised partitioning systems, which in some cases use a mechanism that show striking similarity to eukaryotic DNA segregation. Overall, there have been multiple systems implicated in the dynamic transport of DNA cargo to a new cellular position during the cell cycle but most seem to share a common initial DNA partitioning step, involving the formation of a nucleoprotein complex called the segrosome. The particular features and complex topologies of individual segrosomes depend on both the nature of the DNA binding protein involved and on the recognized centromeric DNA sequence, both of which vary across systems. The combination of in vivo and in vitro approaches, with structural biology has significantly furthered our understanding of the mechanisms underlying DNA trafficking in bacteria. Here, I discuss recent advances and the molecular details of the DNA segregation machinery, focusing on the formation of the segrosome complex.

  16. Modeling bacterial population growth from stochastic single-cell dynamics.

    Science.gov (United States)

    Alonso, Antonio A; Molina, Ignacio; Theodoropoulos, Constantinos

    2014-09-01

    A few bacterial cells may be sufficient to produce a food-borne illness outbreak, provided that they are capable of adapting and proliferating on a food matrix. This is why any quantitative health risk assessment policy must incorporate methods to accurately predict the growth of bacterial populations from a small number of pathogens. In this aim, mathematical models have become a powerful tool. Unfortunately, at low cell concentrations, standard deterministic models fail to predict the fate of the population, essentially because the heterogeneity between individuals becomes relevant. In this work, a stochastic differential equation (SDE) model is proposed to describe variability within single-cell growth and division and to simulate population growth from a given initial number of individuals. We provide evidence of the model ability to explain the observed distributions of times to division, including the lag time produced by the adaptation to the environment, by comparing model predictions with experiments from the literature for Escherichia coli, Listeria innocua, and Salmonella enterica. The model is shown to accurately predict experimental growth population dynamics for both small and large microbial populations. The use of stochastic models for the estimation of parameters to successfully fit experimental data is a particularly challenging problem. For instance, if Monte Carlo methods are employed to model the required distributions of times to division, the parameter estimation problem can become numerically intractable. We overcame this limitation by converting the stochastic description to a partial differential equation (backward Kolmogorov) instead, which relates to the distribution of division times. Contrary to previous stochastic formulations based on random parameters, the present model is capable of explaining the variability observed in populations that result from the growth of a small number of initial cells as well as the lack of it compared to

  17. Fate of deposited cells in an aerobic binary bacterial biofilm

    International Nuclear Information System (INIS)

    Banks, M.K.

    1989-01-01

    A biofilm is a matrix of microbial cells and their extracellular products that is associated with a solid surface. Previous studies on biofilm development have employed only dissolved compounds as growth limiting substrates, without the influence of microbial species invading from the bulk liquid. The goal of this research project was to quantify the kinetics of processes governing suspended biomass turnover in biofilm systems, and the accompanying effects of suspended cell deposition on biofilm population dynamics. Experiments were conducted with two species of bacteria, Pseudomonas putida ATCC 11172 grown on glucose, and Hyphomicrobium ZV620 grown on methanol. Cryptic growth and particulate hydrolysis studies were evaluated, using combinations of these two bacteria, by measuring the uptake of radiolabelled cell lysis products, under batch conditions. Biofilms studies were performed to investigate bacterial deposition, continual biofilm removal by shear induced erosion, and biofilm ecology. Biofilms were developed in a flow cell reactor, under laminar flow conditions. Bacterial species were differentiated by radioactively labelling each species with their carbon substrate. A mathematical model was developed to predict the biofilm ecology of mixed cultures. The equations developed predict biofilm accumulation, as well as substrate and oxygen consumption. Results indicate that cryptic growth will occur for bacteria growing on their own species soluble lysis products and in some cases, bacteria growing on the soluble lysis products of other species. Particulate hydrolysis only occurred for Pseudomonas putida growing on Pseudomonas putida lysis products, but the lack of particulate hydrolysis occurring in the other studies may have been due to the short experimental period

  18. Novel chemically modified bacterial cellulose nanocomposite as potential biomaterial for stem cell therapy applications.

    Science.gov (United States)

    Xavier Acasigua, Gerson Arisoly; de Olyveira, Gabriel Molina; Manzine Costa, Ligia Maria; Braghirolli, Daikelly Iglesias; Medeiros Fossati, Anna Christina; Guastaldi, Antonio Carlos; Pranke, Patricia; Daltro, Gildásio de Cerqueira; Basmaji, Pierre

    2014-03-01

    Bacterial cellulose (BC) has become established as a remarkably versatile biomaterial and can be used in a wide variety of applied scientific applications, especially for medical devices. In this work, the bacterial cellulose fermentation process is modified by the addition of hyaluronic acid and gelatin (1% w/w) to the culture medium before the bacteria is inoculated. Hyaluronic acid and gelatin influence in bacterial cellulose was analyzed using Transmission Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). Adhesion and viability studies with human dental pulp stem cells using natural bacterial cellulose/hyaluronic acid as scaffolds for regenerative medicine are presented for the first time in this work. MTT viability assays show higher cell adhesion in bacterial cellulose/gelatin and bacterial cellulose/ hyaluronic acid scaffolds over time with differences due to fiber agglomeration in bacterial cellulose/gelatin. Confocal microscopy images showed that the cell were adhered and well distributed within the fibers in both types of scaffolds.

  19. Probing living bacterial adhesion by single cell force spectroscopy using atomic force microscopy

    DEFF Research Database (Denmark)

    Zeng, Guanghong; Ogaki, Ryosuke; Regina, Viduthalai R.

    be considered. We have therefore developed a simple and versatile method to make single-cell bacterial probes for measuring single cell adhesion with atomic force microscopy (AFM).[1] A single-cell probe was readily made by picking up a bacterial cell from a glass surface using a tipless AFM cantilever coated...... with a commercial cell adhesive CellTakTM. The method was applied to four different bacterial strains, and single-cell adhesion was measured on three surfaces (fresh glass, hydrophilic glass, mica). Attachment to the cantilever was stable during the 2 h of AFM force measurements, and viability was confirmed by Live....../Dead fluorescence staining at the end of each experiment. The adhesion force and final rupture length were dependent on bacterial strains, surfaces properties, and time of contact. The single-cell probe offers control of the cell immobilization, thus holds advantages over the commonly used multi-cell probes where...

  20. Elaborating European Pharmacopoeia monographs for biotherapeutic proteins using substances from a single source.

    Science.gov (United States)

    Buda, M; Wicks, S; Charton, E

    2016-01-01

    For more than twenty years, the European Pharmacopoeia (Ph. Eur.) monographs for biotherapeutic proteins have been elaborated using the multisource approach (Procedure 1), which has led to robust quality standards for many of the first-generation biotherapeutics. In 2008, the Ph. Eur. opened up the way towards an alternative mechanism for the elaboration of monographs (Procedure 4-BIO pilot phase), which is applied to substances still under patent protection, based on a close collaboration with the Innovator company, to ensure a harmonised global standard and strengthen the quality of the upcoming products. This article describes the lessons learned during the P4-BIO pilot phase and addresses the current thinking on monograph elaboration in the field of biotherapeutics. Case studies are described to illustrate the standardisation challenges associated with the complexity of biotherapeutics and of analytical procedures, as well as the approaches that help ensure expectations are met when setting monograph specifications and allow for compatibility with the development of biosimilars. Emphasis is put on monograph flexibility, notably by including tests that measure process-dependent microheterogeneity (e.g. glycosylation) in the Production section of the monograph. The European Pharmacopoeia successfully concluded the pilot phase of the P4-BIO during its 156 th session on 22-23 November 2016.

  1. The sweet spot for biologics: recent advances in characterization of biotherapeutic glycoproteins.

    Science.gov (United States)

    O'Flaherty, Róisín; Trbojević-Akmačić, Irena; Greville, Gordon; Rudd, Pauline M; Lauc, Gordan

    2018-01-01

    Glycosylation is recognized as a Critical Quality Attribute for therapeutic glycoproteins such as monoclonal antibodies, fusion proteins and therapeutic replacement enzymes. Hence, efficient and quantitative glycan analysis techniques have been increasingly important for their discovery, development and quality control. The aim of this review is to highlight relevant and recent advances in analytical technologies for characterization of biotherapeutic glycoproteins. Areas covered: The review gives an overview of the glycosylation trends of biotherapeutics approved in 2016 and 2017 by FDA. It describes current and novel analytical technologies for characterization of therapeutic glycoproteins and is explored in the context of released glycan, glycopeptide or intact glycoprotein analysis. Ultra performance liquid chromatography, mass spectrometry and capillary electrophoresis technologies are explored in this context. Expert commentary: There is a need for the biopharmaceutical industry to incorporate novel state of the art analytical technologies into existing and new therapeutic glycoprotein workflows for safer and more efficient biotherapeutics and for the improvement of future biotherapeutic design. Additionally, at present, there is no 'gold-standard' approach to address all the regulatory requirements and as such this will involve the use of orthogonal glycoanalytical technologies with a view to gain diagnostic information about the therapeutic glycoprotein.

  2. Mutations That Alter the Bacterial Cell Envelope Increase Lipid Production

    Energy Technology Data Exchange (ETDEWEB)

    Lemmer, Kimberly C.; Zhang, Weiping; Langer, Samantha J.; Dohnalkova, Alice C.; Hu, Dehong; Lemke, Rachelle A.; Piotrowski, Jeff S.; Orr, Galya; Noguera, Daniel R.; Donohue, Timothy J.; Ruby, Edward G.

    2017-05-23

    ABSTRACT

    Lipids from microbes offer a promising source of renewable alternatives to petroleum-derived compounds. In particular, oleaginous microbes are of interest because they accumulate a large fraction of their biomass as lipids. In this study, we analyzed genetic changes that alter lipid accumulation inRhodobacter sphaeroides. By screening anR. sphaeroidesTn5mutant library for insertions that increased fatty acid content, we identified 10 high-lipid (HL) mutants for further characterization. These HL mutants exhibited increased sensitivity to drugs that target the bacterial cell envelope and changes in shape, and some had the ability to secrete lipids, with two HL mutants accumulating ~60% of their total lipids extracellularly. When one of the highest-lipid-secreting strains was grown in a fed-batch bioreactor, its lipid content was comparable to that of oleaginous microbes, with the majority of the lipids secreted into the medium. Based on the properties of these HL mutants, we conclude that alterations of the cell envelope are a previously unreported approach to increase microbial lipid production. We also propose that this approach may be combined with knowledge about biosynthetic pathways, in this or other microbes, to increase production of lipids and other chemicals.

    IMPORTANCEThis paper reports on experiments to understand how to increase microbial lipid production. Microbial lipids are often cited as one renewable replacement for petroleum-based fuels and chemicals, but strategies to increase the yield of these compounds are needed to achieve this goal. While lipid biosynthesis is often well understood, increasing yields of these compounds to industrially relevant levels is a challenge, especially since genetic, synthetic biology, or engineering approaches are not feasible in many microbes. We show that altering the bacterial cell envelope can be used to increase

  3. Role of the T cell receptor ligand affinity in T cell activation by bacterial superantigens

    DEFF Research Database (Denmark)

    Andersen, P S; Geisler, C; Buus, S

    2001-01-01

    Similar to native peptide/MHC ligands, bacterial superantigens have been found to bind with low affinity to the T cell receptor (TCR). It has been hypothesized that low ligand affinity is required to allow optimal TCR signaling. To test this, we generated variants of Staphylococcus enterotoxin C3...

  4. Pre-existing biotherapeutic-reactive antibodies: survey results within the American Association of Pharmaceutical Scientists.

    Science.gov (United States)

    Xue, Li; Fiscella, Michele; Rajadhyaksha, Manoj; Goyal, Jaya; Holland, Claire; Gorovits, Boris; Morimoto, Alyssa

    2013-07-01

    The immunogenicity profile of a biotherapeutic is determined by a multitude of product and patient-related risk factors that can influence the observed incidence and clinical consequences of immunogenicity. Pre-existing antibodies, i.e., biotherapeutic-reactive antibodies present in samples from treatment-naïve subjects, have been commonly observed during immunogenicity assessments; however their relevance in terms of the safety and efficacy of a biotherapeutic is poorly understood. An American Association of Pharmaceutical Scientists-sponsored survey was conducted to gather information about the prevalence, nature, and consequences of pre-existing antibodies in clinical and nonclinical studies. The survey results indicate that pre-existing antibodies against a variety of biotherapeutics (e.g., mAbs, fusion proteins) are frequently encountered, especially in the context of autoimmune diseases, but that the methods and approaches used to detect, characterize, and report these antibodies vary. In most cases, pre-existing antibodies did not appear to have clinical consequences; however, a few of the respondents reported having observed an effect on pharmacokinetic, pharmacodynamic, safety, and/or efficacy parameters. The findings from this survey are an important first step in evaluating the potential risks associated with the presence of pre-existing antibodies and highlight the importance of standardizing the approaches for detection and characterization of these antibodies. Cross-industry sharing of case studies and relevant data collection will help better inform biotherapeutic risk/benefit profiles and provide deeper understanding of the biological consequences of pre-existing antibodies.

  5. Heterotrophic free-living and particle-bound bacterial cell size in the ...

    Indian Academy of Sciences (India)

    PRAKASH

    bacterial cell size was similar in all the five water courses, different sets of environmental variables apparently control the heterotrophic bacterial cell size ... major force controlling both the morphological and the taxonomic structure of ...... 18th edition, pp1–1000. Bennet S J, Sanders R W and Porter K G 1990 Heterotrophic,.

  6. Mechanism of cell integration on biomaterial implant surfaces in the presence of bacterial contamination

    NARCIS (Netherlands)

    Yue, Chongxia; van der Mei, Henny C.; Kuijer, Roel; Busscher, Henk J.; Rochford, Edward T. J.

    2015-01-01

    Bacterial contamination during biomaterial implantation is often unavoidable, yielding a combat between cells and bacteria. Here we aim to determine the modulatory function of bacterial components on stem-cell, fibroblast, and osteoblast adhesion to a titanium alloy, including the role of

  7. Comparison of the cytotoxic effect of polystyrene latex nanoparticles on planktonic cells and bacterial biofilms

    International Nuclear Information System (INIS)

    Nomura, Toshiyuki; Fujisawa, Eri; Itoh, Shikibu; Konishi, Yasuhiro

    2016-01-01

    The cytotoxic effect of positively charged polystyrene latex nanoparticles (PSL NPs) was compared between planktonic bacterial cells and bacterial biofilms using confocal laser scanning microscopy, atomic force microscopy, and a colony counting method. Pseudomonas fluorescens, which is commonly used in biofilm studies, was employed as the model bacteria. We found that the negatively charged bacterial surface of the planktonic cells was almost completely covered with positively charged PSL NPs, leading to cell death, as indicated by the NP concentration being greater than that required to achieve single layer coverage. In addition, the relationship between surface coverage and cell viability of P. fluorescens cells correlated well with the findings in other bacterial cells (Escherichia coli and Lactococcuslactis). However, most of the bacterial cells that formed the biofilm were viable despite the positively charged PSL NPs being highly toxic to planktonic bacterial cells. This indicated that bacterial cells embedded in the biofilm were protected by self-produced extracellular polymeric substances (EPS) that provide resistance to antibacterial agents. In conclusion, mature biofilms covered with EPS exhibit resistance to NP toxicity as well as antibacterial agents.

  8. Comparison of the cytotoxic effect of polystyrene latex nanoparticles on planktonic cells and bacterial biofilms

    Energy Technology Data Exchange (ETDEWEB)

    Nomura, Toshiyuki, E-mail: nomura@chemeng.osakafu-u.ac.jp; Fujisawa, Eri; Itoh, Shikibu; Konishi, Yasuhiro [Osaka Prefecture University, Department of Chemical Engineering (Japan)

    2016-06-15

    The cytotoxic effect of positively charged polystyrene latex nanoparticles (PSL NPs) was compared between planktonic bacterial cells and bacterial biofilms using confocal laser scanning microscopy, atomic force microscopy, and a colony counting method. Pseudomonas fluorescens, which is commonly used in biofilm studies, was employed as the model bacteria. We found that the negatively charged bacterial surface of the planktonic cells was almost completely covered with positively charged PSL NPs, leading to cell death, as indicated by the NP concentration being greater than that required to achieve single layer coverage. In addition, the relationship between surface coverage and cell viability of P. fluorescens cells correlated well with the findings in other bacterial cells (Escherichia coli and Lactococcuslactis). However, most of the bacterial cells that formed the biofilm were viable despite the positively charged PSL NPs being highly toxic to planktonic bacterial cells. This indicated that bacterial cells embedded in the biofilm were protected by self-produced extracellular polymeric substances (EPS) that provide resistance to antibacterial agents. In conclusion, mature biofilms covered with EPS exhibit resistance to NP toxicity as well as antibacterial agents.

  9. Comparison of the cytotoxic effect of polystyrene latex nanoparticles on planktonic cells and bacterial biofilms

    Science.gov (United States)

    Nomura, Toshiyuki; Fujisawa, Eri; Itoh, Shikibu; Konishi, Yasuhiro

    2016-06-01

    The cytotoxic effect of positively charged polystyrene latex nanoparticles (PSL NPs) was compared between planktonic bacterial cells and bacterial biofilms using confocal laser scanning microscopy, atomic force microscopy, and a colony counting method. Pseudomonas fluorescens, which is commonly used in biofilm studies, was employed as the model bacteria. We found that the negatively charged bacterial surface of the planktonic cells was almost completely covered with positively charged PSL NPs, leading to cell death, as indicated by the NP concentration being greater than that required to achieve single layer coverage. In addition, the relationship between surface coverage and cell viability of P. fluorescens cells correlated well with the findings in other bacterial cells ( Escherichia coli and Lactococcus lactis). However, most of the bacterial cells that formed the biofilm were viable despite the positively charged PSL NPs being highly toxic to planktonic bacterial cells. This indicated that bacterial cells embedded in the biofilm were protected by self-produced extracellular polymeric substances (EPS) that provide resistance to antibacterial agents. In conclusion, mature biofilms covered with EPS exhibit resistance to NP toxicity as well as antibacterial agents.

  10. [Genetic transformation and fate of heterological DNA in bacterial cells].

    Science.gov (United States)

    Piechowska, Mirosława

    2015-01-01

    Secretion of a metabolite enabling Streptococci to undergo genetic transformation was discovered. The metabolite combined with an optimization process were applied to increase the transformation yield about 20-fold. It was observed that large amounts of DNA exert a bactericidal effect, indicating the ability of at least 70% of cells to uptake the polymer. While studying the molecular mechanism of transformation of Bacillus subtilis it was shown that the uptaken DNA forms complexes with bacterial proteins, which hinders determination of its structure. A method was found to dissociate these complexes which enabled to determine the single-stranded structure of the uptaken DNA. Donor DNA fragments incorporated into the host DNA were of about 10 Da. Non-transforming DNA can be uptaken similarly but does not undergo incorporation into the host DNA. The selectivity of Bacillus subtilis receptors was determined towards DNA of phages containing modified bases: uracil, putrescinyl-thymine and its acetylated derivative, 5'-hydroxymethylcytosine and its glycosylated derivative and also towards double-stranded RNA of f2 phage. All these modifications were tolerated by the cellular receptors, with the exception of glycosylation and the 2'-OH group in RNA.

  11. Nonmalignant T cells stimulate growth of T-cell lymphoma cells in the presence of bacterial toxins

    DEFF Research Database (Denmark)

    Woetmann, Anders; Lovato, Paola; Eriksen, Karsten W

    2007-01-01

    Bacterial toxins including staphylococcal enterotoxins (SEs) have been implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCLs). Here, we investigate SE-mediated interactions between nonmalignant T cells and malignant T-cell lines established from skin and blood of CTCL patients....... The malignant CTCL cells express MHC class II molecules that are high-affinity receptors for SE. Although treatment with SE has no direct effect on the growth of the malignant CTCL cells, the SE-treated CTCL cells induce vigorous proliferation of the SE-responsive nonmalignant T cells. In turn, the nonmalignant...... T cells enhance proliferation of the malignant cells in an SE- and MHC class II-dependent manner. Furthermore, SE and, in addition, alloantigen presentation by malignant CTCL cells to irradiated nonmalignant CD4(+) T-cell lines also enhance proliferation of the malignant cells. The growth...

  12. Bacterial Colonization of Host Cells in the Absence of Cholesterol

    Science.gov (United States)

    Gilk, Stacey D.; Cockrell, Diane C.; Luterbach, Courtney; Hansen, Bryan; Knodler, Leigh A.; Ibarra, J. Antonio; Steele-Mortimer, Olivia; Heinzen, Robert A.

    2013-01-01

    Reports implicating important roles for cholesterol and cholesterol-rich lipid rafts in host-pathogen interactions have largely employed sterol sequestering agents and biosynthesis inhibitors. Because the pleiotropic effects of these compounds can complicate experimental interpretation, we developed a new model system to investigate cholesterol requirements in pathogen infection utilizing DHCR24−/− mouse embryonic fibroblasts (MEFs). DHCR24−/− MEFs lack the Δ24 sterol reductase required for the final enzymatic step in cholesterol biosynthesis, and consequently accumulate desmosterol into cellular membranes. Defective lipid raft function by DHCR24−/− MEFs adapted to growth in cholesterol-free medium was confirmed by showing deficient uptake of cholera-toxin B and impaired signaling by epidermal growth factor. Infection in the absence of cholesterol was then investigated for three intracellular bacterial pathogens: Coxiella burnetii, Salmonella enterica serovar Typhimurium, and Chlamydia trachomatis. Invasion by S. Typhimurium and C. trachomatis was unaltered in DHCR24−/− MEFs. In contrast, C. burnetii entry was significantly decreased in −cholesterol MEFs, and also in +cholesterol MEFs when lipid raft-associated αVβ3 integrin was blocked, suggesting a role for lipid rafts in C. burnetii uptake. Once internalized, all three pathogens established their respective vacuolar niches and replicated normally. However, the C. burnetii-occupied vacuole within DHCR24−/− MEFs lacked the CD63-postive material and multilamellar membranes typical of vacuoles formed in wild type cells, indicating cholesterol functions in trafficking of multivesicular bodies to the pathogen vacuole. These data demonstrate that cholesterol is not essential for invasion and intracellular replication by S. Typhimurium and C. trachomatis, but plays a role in C. burnetii-host cell interactions. PMID:23358892

  13. Chemoporation using saponins or cholates: an alternative method for transformation of bacterial cells.

    Science.gov (United States)

    Ravnikar, Matjaz; Irman, Andreja; Radić, Natasa; Lunder, Mojca; Strukelj, Borut

    2009-12-01

    A new method for fast transformation of competent bacterial cells has been developed. The transformation is induced with cholic acid analogues or saponins which cause reversible disruption of the bacterial membrane. This method shortens the time of transformation without significant loss of transformation efficiency in comparison to heat shock method and is the first reported chemically-induced transformation. New data about interactions between cholates and biomembranes is revealed that may contribute to better understanding of bacterial transformation.

  14. Chemically synthesized silver nanoparticles as cell lysis agent for bacterial genomic DNA isolation

    Science.gov (United States)

    Goswami, Gunajit; Boruah, Himangshu; Gautom, Trishnamoni; Jyoti Hazarika, Dibya; Barooah, Madhumita; Boro, Robin Chandra

    2017-12-01

    Silver nanoparticles (AgNPs) have seen a recent spurt of use in varied fields of science. In this paper, we showed a novel application of AgNP as a promising microbial cell-lysis agent for genomic DNA isolation. We utilized chemically synthesized AgNPs for lysing bacterial cells to isolate their genomic DNA. The AgNPs efficiently lysed bacterial cells to yield good quality DNA that could be subsequently used for several molecular biology works.

  15. Microfabrication of Si and GaAs by Plasma Etching Process Using Bacterial Cells as an Etching Mask Material

    Science.gov (United States)

    Matsutani, Akihiro; Takada, Ayako

    2012-08-01

    We demonstrated that bacterial cells can be used as a mask material for microfabrication of GaAs and Si by a Cl2 inductively coupled plasma (ICP) etching process. The etching rate of Escherichia coli cells was similar to that of electron beam resist or nanoimprint resist. We also demonstrated the degradation of bacterial cells by low-pressure plasma treatment using O2, Ar, air, and H2O for removal of bacterial cells as the etching mask material. Bacterial cells were efficiently degraded by ions in the low-pressure discharge plasma. The proposed process using bacterial cells can be expected to be applied to semiconductor dry etching processes.

  16. A newly isolated probiotic Enterococcus faecalis strain from vagina microbiota enhances apoptosis of human cancer cells.

    Science.gov (United States)

    Nami, Y; Abdullah, N; Haghshenas, B; Radiah, D; Rosli, R; Yari Khosroushahi, A

    2014-08-01

    This study aimed to describe probiotic properties and bio-therapeutic effects of newly isolated Enterococcus faecalis from the human vaginal tract. The Enterococcus faecalis strain was originally isolated from the vaginal microbiota of Iranian women and was molecularly identified using 16SrDNA gene sequencing. Some biochemical methodologies were preliminarily used to characterize the probiotic potential of Ent. faecalis, including antibiotic susceptibility, antimicrobial activity, as well as acid and bile resistance. The bio-therapeutic effects of this strain's secreted metabolites on four human cancer cell lines (AGS, HeLa, MCF-7 and HT-29) and one normal cell line (HUVEC) were evaluated by cytotoxicity assay and apoptosis scrutiny. The characterization results demonstrated into the isolated bacteria strain revealed probiotic properties, such as antibiotic susceptibility, antimicrobial activity and resistance under conditions similar to those in the gastrointestinal tract. Results of bio-therapeutic efficacy assessments illustrated acceptable apoptotic effects on four human cancer cell lines and negligible side effects on assayed normal cell line. Our findings revealed that the apoptotic effect of secreted metabolites mainly depended on proteins secreted by Ent. faecalis on different cancer cells. These proteins can induce the apoptosis of cancer cells. The metabolites produced by this vaginal Ent. faecalis strain can be used as alternative pharmaceutical compounds with promising therapeutic indices because they are not cytotoxic to normal mammalian cells. Accordingly, the physicochemical, structural and functional properties of the secreted anticancer substances should be further investigated before using them as anticancer therapeutics. This study aim to screen total bacterial secreted metabolites as a wealthy source to find the new active compounds to introduce as anticancer therapeutics in the future. © 2014 The Society for Applied Microbiology.

  17. Probing living bacterial adhesion by single cell force spectroscopy using atomic force microscopy

    DEFF Research Database (Denmark)

    Zeng, Guanghong; Ogaki, Ryosuke; Regina, Viduthalai R.

    Bacteria initiate attachment to the surfaces with the aid of different extracellular polymers. To quantitatively study how these polymers mediate bacterial adhesion and possibly their interactions, it is essential to go down to single cell level, with in mind that cell-to-cell variation should...... with a commercial cell adhesive CellTakTM. The method was applied to four different bacterial strains, and single-cell adhesion was measured on three surfaces (fresh glass, hydrophilic glass, mica). Attachment to the cantilever was stable during the 2 h of AFM force measurements, and viability was confirmed by Live...

  18. Heterotrophic free-living and particle-bound bacterial cell size in the ...

    Indian Academy of Sciences (India)

    Regression analysis revealed that 18% of the variation in mean heterotrophic free-living bacterial cell size was due to biological oxygen demand (BOD) in the river Arkavathy, 11% due to surface water velocity (SWV) in the river Cauvery and 11% due to temperature in the river Kapila. Heterotrophic particle-bound bacterial ...

  19. A Consensus Genome-scale Reconstruction of Chinese Hamster Ovary Cell Metabolism

    DEFF Research Database (Denmark)

    Hefzi, Hooman; Ang, Kok Siong; Hanscho, Michael

    2016-01-01

    Chinese hamster ovary (CHO) cells dominate biotherapeutic protein production and are widely used in mammalian cell line engineering research. To elucidate metabolic bottlenecks in protein production and to guide cell engineering and bioprocess optimization, we reconstructed the metabolic pathways...

  20. Harnessing cell-to-cell variations to probe bacterial structure and biophysics

    Science.gov (United States)

    Cass, Julie A.

    Advances in microscopy and biotechnology have given us novel insights into cellular biology and physics. While bacteria were long considered to be relatively unstructured, the development of fluorescence microscopy techniques, and spatially and temporally resolved high-throughput quantitative studies, have uncovered that the bacterial cell is highly organized, and its structure rigorously maintained. In this thesis I will describe our gateTool software, designed to harness cell-to-cell variations to probe bacterial structure, and discuss two exciting aspects of structure that we have employed gateTool to investigate: (i) chromosome organization and the cellular mechanisms for controlling DNA dynamics, and (ii) the study of cell wall synthesis, and how the genes in the synthesis pathway impact cellular shape. In the first project, we develop a spatial and temporal mapping of cell-cycle-dependent chromosomal organization, and use this quantitative map to discover that chromosomal loci segregate from midcell with universal dynamics. In the second project, I describe preliminary time- lapse and snapshot imaging analysis suggesting phentoypical coherence across peptidoglycan synthesis pathways.

  1. Metal ion leachates and the physico-chemical stability of biotherapeutic drug products.

    Science.gov (United States)

    Kumar, Sandeep; Zhou, Shuxia; Singh, Satish K

    2014-01-01

    Metal ions that leach into biotherapeutic drug product solution during manufacturing and storage, result in contamination that can cause physico-chemical degradation of the active molecule. In this review, we describe various mechanisms by which metal ion leachates can interact with therapeutic proteins and antibodies. Site-specific modifications due to metal catalyzed oxidation (MCO) of the therapeutic proteins cause them to become destabilized and potentially increasingly aggregation prone. We have examined the molecular sequences and structures for three case studies, human relaxin (hRlx), human growth hormone (hGH) and an IgG2 mAb to rationalize the experimental findings related to their MCO. The analysis indicates that metal-binding sites lie in close spatial proximities to predicted aggregation prone regions in these molecules. From the perspective of pharmaceutical development of biotherapeutic drugs, this link between molecular origins of MCO and subsequent aggregation is undesirable. This article further suggests molecular design strategies involving disruption of APRs that may also help mitigate the impact of metal ion leachates on biotherapeutic drug products as well as improving their solubility.

  2. EEVD motif of heat shock cognate protein 70 contributes to bacterial uptake by trophoblast giant cells

    Directory of Open Access Journals (Sweden)

    Kim Suk

    2009-12-01

    Full Text Available Abstract Background The uptake of abortion-inducing pathogens by trophoblast giant (TG cells is a key event in infectious abortion. However, little is known about phagocytic functions of TG cells against the pathogens. Here we show that heat shock cognate protein 70 (Hsc70 contributes to bacterial uptake by TG cells and the EEVD motif of Hsc70 plays an important role in this. Methods Brucella abortus and Listeria monocytogenes were used as the bacterial antigen in this study. Recombinant proteins containing tetratricopeptide repeat (TPR domains were constructed and confirmation of the binding capacity to Hsc70 was assessed by ELISA. The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice. Results The monoclonal antibody that inhibits bacterial uptake by TG cells reacted with the EEVD motif of Hsc70. Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells. Infectious abortion was also prevented by blocking the EEVD motif of Hsc70. Conclusions Our results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif. These molecules may be useful in the development of methods for preventing infectious abortion.

  3. Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-Soo; Torelli, Marco; Hamers, Robert J.; Murphy, Catherine; Orr, Galya; Haynes, Christy L.

    2014-01-01

    A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.

  4. Bacterial vaginosis (clue cell-positive discharge) : diagnostic, ultra-structural and therapeutic aspects

    NARCIS (Netherlands)

    W.I. van der Meijden (Willem)

    1987-01-01

    textabstractThis thesis deals with several aspects of (abnormal) vaginal discharge, focusing especially on clue cell-positive discharge (bacterial vaginosis, nonspecific vaginitis). It reports data on epidemiology and clinical features, pathogenesis, and treatment of this vaginal disease entity,

  5. Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency.

    OpenAIRE

    Tu, Qiang; Yin, Jia; Fu, Jun; Herrmann, Jennifer; Li, Yuezhong; Yin, Yulong; Stewart, A Francis; Müller, Rolf; Zhang, Youming

    2016-01-01

    Bacterial competent cells are essential for cloning, construction of DNA libraries, and mutagenesis in every molecular biology laboratory. Among various transformation methods, electroporation is found to own the best transformation efficiency. Previous electroporation methods are based on washing and electroporating the bacterial cells in ice-cold condition that make them fragile and prone to death. Here we present simple temperature shift based methods that improve DNA transformation and re...

  6. Mechanisms of ion-bombardment-induced DNA transfer into bacterial E. coli cells

    International Nuclear Information System (INIS)

    Yu, L.D.; Sangwijit, K.; Prakrajang, K.; Phanchaisri, B.; Thongkumkoon, P.; Thopan, P.; Singkarat, S.; Anuntalabhochai, S.

    2014-01-01

    Highlights: • Ion bombardment could induce DNA transfer into E. coli cells. • The DNA transfer induction depended on ion energy and fluence. • The mechanism was associated with the bacterial cell envelope structure. • A mechanism phase diagram was proposed to summarize the mechanism. - Abstract: As a useful ion beam biotechnology, ion-bombardment-induced DNA transfer into bacterial Escherichia coli (E. coli) cells has been successfully operated using argon ions. In the process ion bombardment of the bacterial cells modifies the cell envelope materials to favor the exogenous DNA molecules to pass through the envelope to enter the cell. The occurrence of the DNA transfer induction was found ion energy and fluence dependent in a complex manner. At ion energy of a few keV and a few tens of keV to moderate fluences the DNA transfer could be induced by ion bombardment of the bacterial cells, while at the same ion energy but to high fluences DNA transfer could not be induced. On the other hand, when the ion energy was medium, about 10–20 keV, the DNA transfer could not be induced by ion bombardment of the cells. The complexity of the experimental results indicated a complex mechanism which should be related to the complex structure of the bacterial E. coli cell envelope. A phase diagram was proposed to interpret different mechanisms involved as functions of the ion energy and fluence

  7. Bacterial toxins fuel disease progression in cutaneous T-cell lymphoma

    DEFF Research Database (Denmark)

    Willerslev-Olsen, Andreas; Krejsgaard, Thorbjørn; Lindahl, Lise Maria

    2013-01-01

    In patients with cutaneous T-cell lymphoma (CTCL) bacterial infections constitute a major clinical problem caused by compromised skin barrier and a progressive immunodeficiency. Indeed, the majority of patients with advanced disease die from infections with bacteria, e.g., Staphylococcus aureus....... Bacterial toxins such as staphylococcal enterotoxins (SE) have long been suspected to be involved in the pathogenesis in CTCL. Here, we review links between bacterial infections and CTCL with focus on earlier studies addressing a direct role of SE on malignant T cells and recent data indicating novel...

  8. A Culture-Independent Approach to Enrich Endophytic Bacterial Cells from Sugarcane Stems for Community Characterization.

    Science.gov (United States)

    Dos-Santos, Carlos M; de Souza, Daniel G; Balsanelli, Eduardo; Cruz, Leonardo Magalhães; de Souza, Emanuel M; Baldani, José I; Schwab, Stefan

    2017-08-01

    Bacterial endophytes constitute a very diverse community and they confer important benefits which help to improve agricultural yield. Some of these benefits remain underexplored or little understood, mainly due to the bottlenecks associated with the plant feature, a low number of endophytic bacterial cells in relation to the plant, and difficulties in accessing these bacteria using cultivation-independent methods. Enriching endophytic bacterial cells from plant tissues, based on a non-biased, cultivation-independent physical enrichment method, may help to circumvent those problems, especially in the case of sugarcane stems, which have a high degree of interfering factors, such as polysaccharides, phenolic compounds, nucleases, and fibers. In the present study, an enrichment approach for endophytic bacterial cells from sugarcane lower stems is described. The results demonstrate that the enriched bacterial cells are suitable for endophytic community characterization. A community analysis revealed the presence of previously well-described but also novel endophytic bacteria in sugarcane tissues which may exert functions such as plant growth promotion and biological control, with a predominance of the Proteobacterial phylum, but also Actinobacteria, Bacteroidetes, and Firmicutes, among others. In addition, by comparing the present and literature data, it was possible to list the most frequently detected bacterial endophyte genera in sugarcane tissues. The presented enrichment approach paves the way for improved future research toward the assessment of endophytic bacterial community in sugarcane and other biofuel crops.

  9. Mechanosensitive channels and bacterial cell wall integrity: Does life end with a bang or a whimper?

    NARCIS (Netherlands)

    M. Reuter (Marcel); N.J. Hayward (Nicholas); S.S. Black (Susan); S. Miller (Samantha); D.T.F. Dryden (David); I.R. Booth (Ian)

    2014-01-01

    textabstractMechanogated channels are fundamental components of bacterial cells that enable retention of physical integrity during extreme increases in cell turgor. Optical tweezers combined with microfluidics have been used to study the fate of individual Escherichia coli cells lacking such

  10. Mechanism of cell integration on biomaterial implant surfaces in the presence of bacterial contamination.

    Science.gov (United States)

    Yue, Chongxia; van der Mei, Henny C; Kuijer, Roel; Busscher, Henk J; Rochford, Edward T J

    2015-11-01

    Bacterial contamination during biomaterial implantation is often unavoidable, yielding a combat between cells and bacteria. Here we aim to determine the modulatory function of bacterial components on stem-cell, fibroblast, and osteoblast adhesion to a titanium alloy, including the role of toll-like-receptors (TLRs). Presence of heat-sacrificed Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, or Pseudomonas aeruginosa induced dose and cell-type dependent responses. Stem-cells were most sensitive to bacterial presence, demonstrating decreased adhesion number yet increased adhesion effort with a relatively large focal adhesion contact area. Blocking TLRs had no effect on stem-cell adhesion in presence of S. aureus, but blocking both TLR2 and TLR4 induced an increased adhesion effort in presence of E. coli. Neither lipopolysaccharide, lipoteichoic acid, nor bacterial DNA provoked the same cell response as did whole bacteria. Herewith we suggest a new mechanism as to how biomaterials are integrated by cells despite the unavoidable presence of bacterial contamination. Stimulation of host cell integration of implant surfaces may open a new window to design new biomaterials with enhanced healing, thereby reducing the risk of biomaterial-associated infection of both "hardware-based" implants as well as of tissue-engineered constructs, known to suffer from similarly high infection risks as currently prevailing in "hardware-based" implants. © 2015 Wiley Periodicals, Inc.

  11. The DSF Family of Cell-Cell Signals: An Expanding Class of Bacterial Virulence Regulators.

    Directory of Open Access Journals (Sweden)

    Robert P Ryan

    2015-07-01

    Full Text Available Many pathogenic bacteria use cell-cell signaling systems involving the synthesis and perception of diffusible signal molecules to control virulence as a response to cell density or confinement to niches. Bacteria produce signals of diverse structural classes. Signal molecules of the diffusible signal factor (DSF family are cis-2-unsaturated fatty acids. The paradigm is cis-11-methyl-2-dodecenoic acid from Xanthomonas campestris pv. campestris (Xcc, which controls virulence in this plant pathogen. Although DSF synthesis was thought to be restricted to the xanthomonads, it is now known that structurally related molecules are produced by the unrelated bacteria Burkholderia cenocepacia and Pseudomonas aeruginosa. Furthermore, signaling involving these DSF family members contributes to bacterial virulence, formation of biofilms and antibiotic tolerance in these important human pathogens. Here we review the recent advances in understanding DSF signaling and its regulatory role in different bacteria. These advances include the description of the pathway/mechanism of DSF biosynthesis, identification of novel DSF synthases and new members of the DSF family, the demonstration of a diversity of DSF sensors to include proteins with a Per-Arnt-Sim (PAS domain and the description of some of the signal transduction mechanisms that impinge on virulence factor expression. In addition, we address the role of DSF family signals in interspecies signaling that modulates the behavior of other microorganisms. Finally, we consider a number of recently reported approaches for the control of bacterial virulence through the modulation of DSF signaling.

  12. Analysis of Bacterial Cell Surface Chemical Composition Using Cryogenic X-Ray Photoelectron Spectroscopy.

    Science.gov (United States)

    Ramstedt, Madeleine; Shchukarev, Andrey

    2016-01-01

    This chapter describes a method for measuring the average surface chemical composition with respect to lipids, polysaccharides, and peptides (protein + peptidoglycan) for the outer part of the bacterial cell wall. Bacterial cultures grown over night are washed with a buffer or saline at controlled pH. The analysis is done on fast-frozen bacterial cell pellets obtained after centrifugation, and the analysis requires access to X-ray photoelectron spectroscopy instrumentation that can perform analyses at cryogenic temperatures (for example using liquid nitrogen). The method can be used to monitor changes in the cell wall composition following environmental stimuli or genetic mutations. The data obtained originate from the outermost part of the cell wall. Thus, it is expected that for gram-negative bacteria only the outer membrane and part of the periplasmic peptidoglycan layer is probed during analysis, and for gram-positive bacteria only the top nanometers of the peptidoglycan layer of the cell wall is monitored.

  13. Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR.

    Science.gov (United States)

    Romaniuk, Joseph A H; Cegelski, Lynette

    2015-10-05

    The ability to characterize bacterial cell-wall composition and structure is crucial to understanding the function of the bacterial cell wall, determining drug modes of action and developing new-generation therapeutics. Solid-state NMR has emerged as a powerful tool to quantify chemical composition and to map cell-wall architecture in bacteria and plants, even in the context of unperturbed intact whole cells. In this review, we discuss solid-state NMR approaches to define peptidoglycan composition and to characterize the modes of action of old and new antibiotics, focusing on examples in Staphylococcus aureus. We provide perspectives regarding the selected NMR strategies as we describe the exciting and still-developing cell-wall and whole-cell NMR toolkit. We also discuss specific discoveries regarding the modes of action of vancomycin analogues, including oritavancin, and briefly address the reconsideration of the killing action of β-lactam antibiotics. In such chemical genetics approaches, there is still much to be learned from perturbations enacted by cell-wall assembly inhibitors, and solid-state NMR approaches are poised to address questions of cell-wall composition and assembly in S. aureus and other organisms. © 2015 The Author(s).

  14. Multiscale modeling of bacterial colonies: how pili mediate the dynamics of single cells and cellular aggregates

    Science.gov (United States)

    Pönisch, Wolfram; Weber, Christoph A.; Juckeland, Guido; Biais, Nicolas; Zaburdaev, Vasily

    2017-01-01

    Neisseria gonorrhoeae is the causative agent of one of the most common sexually transmitted diseases, gonorrhea. Over the past two decades there has been an alarming increase of reported gonorrhea cases where the bacteria were resistant to the most commonly used antibiotics thus prompting for alternative antimicrobial treatment strategies. The crucial step in this and many other bacterial infections is the formation of microcolonies, agglomerates consisting of up to several thousands of cells. The attachment and motility of cells on solid substrates as well as the cell-cell interactions are primarily mediated by type IV pili, long polymeric filaments protruding from the surface of cells. While the crucial role of pili in the assembly of microcolonies has been well recognized, the exact mechanisms of how they govern the formation and dynamics of microcolonies are still poorly understood. Here, we present a computational model of individual cells with explicit pili dynamics, force generation and pili-pili interactions. We employ the model to study a wide range of biological processes, such as the motility of individual cells on a surface, the heterogeneous cell motility within the large cell aggregates, and the merging dynamics and the self-assembly of microcolonies. The results of numerical simulations highlight the central role of pili generated forces in the formation of bacterial colonies and are in agreement with the available experimental observations. The model can quantify the behavior of multicellular bacterial colonies on biologically relevant temporal and spatial scales and can be easily adjusted to include the geometry and pili characteristics of various bacterial species. Ultimately, the combination of the microbiological experimental approach with the in silico model of bacterial colonies might provide new qualitative and quantitative insights on the development of bacterial infections and thus pave the way to new antimicrobial treatments.

  15. Live cell imaging of bacterial cells: Pyrenoylpyrrole-based fluorescence labeling.

    Science.gov (United States)

    Arun Divakar, Mathiyazhagan; Shanmugam, Sivakumar

    2017-10-01

    A novel substituted pyrenoylpyrroles was synthesized by the reaction of pyrenoyl chalcone, TosMIC and methyl iodide under mild condition. All the synthesized compounds were screened for their bioactivity, and the MIC was determined, among which few compounds showed moderate antibacterial activity toward Gram-positive as well as Gram-negative bacteria. Further, cytotoxicity assay ascertained that these compounds were non-toxic to mammalian cells as well. The pyrene chromophore in the synthesized compounds (3a-e) and (5a-e) is responsible for the good photophysical properties which have an absorbance at λ 340 nm and emission at λ 410 nm. Hence, two of the selected novel synthesized compounds with non-cytotoxic nature prospected for bio-imaging of bacterial cells using high-content screening analysis show that the molecule is suitable for microbial imaging in pathological diagnostic studies. © 2017 John Wiley & Sons A/S.

  16. Bacterial Cell Growth Inhibitors Targeting Undecaprenyl Diphosphate Synthase and Undecaprenyl Diphosphate Phosphatase.

    Science.gov (United States)

    Wang, Yang; Desai, Janish; Zhang, Yonghui; Malwal, Satish R; Shin, Christopher J; Feng, Xinxin; Sun, Hong; Liu, Guizhi; Guo, Rey-Ting; Oldfield, Eric

    2016-10-19

    We synthesized a series of benzoic acids and phenylphosphonic acids and investigated their effects on the growth of Staphylococcus aureus and Bacillus subtilis. One of the most active compounds, 5-fluoro-2-(3-(octyloxy)benzamido)benzoic acid (7, ED 50 ∼0.15 μg mL -1 ) acted synergistically with seven antibiotics known to target bacterial cell-wall biosynthesis (a fractional inhibitory concentration index (FICI) of ∼0.35, on average) but had indifferent effects in combinations with six non-cell-wall biosynthesis inhibitors (average FICI∼1.45). The most active compounds were found to inhibit two enzymes involved in isoprenoid/bacterial cell-wall biosynthesis: undecaprenyl diphosphate synthase (UPPS) and undecaprenyl diphosphate phosphatase (UPPP), but not farnesyl diphosphate synthase, and there were good correlations between bacterial cell growth inhibition, UPPS inhibition, and UPPP inhibition. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. The innate immune protein Nod2 binds directly to MDP, a bacterial cell wall fragment.

    Science.gov (United States)

    Grimes, Catherine Leimkuhler; Ariyananda, Lushanti De Zoysa; Melnyk, James E; O'Shea, Erin K

    2012-08-22

    Mammalian Nod2 is an intracellular protein that is implicated in the innate immune response to the bacterial cell wall and is associated with the development of Crohn's disease, Blau syndrome, and gastrointestinal cancers. Nod2 is required for an immune response to muramyl dipeptide (MDP), an immunostimulatory fragment of bacterial cell wall, but it is not known whether MDP binds directly to Nod2. We report the expression and purification of human Nod2 from insect cells. Using novel MDP self-assembled monolayers (SAMs), we provide the first biochemical evidence for a direct, high-affinity interaction between Nod2 and MDP.

  18. A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

    Science.gov (United States)

    Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman

    2014-07-01

    The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Use of polymeric membranes for purification of an E. coli expressed biotherapeutic protein.

    Science.gov (United States)

    Muthukumar, S; Rathore, Anurag S

    2016-01-01

    Polymers have had a significant impact on the field of bioseparations in the past few decades. Most recently, membrane chromatography has emerged as an efficient alternative to the conventional packed-bed chromatography by eliminating the diffusion-related limitations associated with the traditional resin beads. In this article, we examine six membrane adsorbers for purification of granulocyte colony-stimulating factor (GCSF), an Escherichia coli-based biotherapeutic. These adsorbers differ either in their base matrix or in the surface chemistry. The role of interactions between the filter surfaces and the protein molecules in effecting these separations is the focus of the article.

  20. Evaluation of the sensitivity of bacterial and yeast cells to cold atmospheric plasma jet treatments.

    Science.gov (United States)

    Sharkey, Michael A; Chebbi, Ahmed; McDonnell, Kevin A; Staunton, Claire; Dowling, Denis P

    2015-06-07

    The focus of this research was first to determine the influence of the atmospheric plasma drive frequency on the generation of atomic oxygen species and its correlation with the reduction of bacterial load after treatment in vitro. The treatments were carried out using a helium-plasma jet source called PlasmaStream™. The susceptibility of multiple microbial cell lines was investigated in order to compare the response of gram-positive and gram-negative bacteria, as well as a yeast cell line to the atmospheric plasma treatment. It was observed for the source evaluated that at a frequency of 160 kHz, increased levels of oxygen-laden active species (i.e., OH, NO) were generated. At this frequency, the maximum level of bacterial inactivation in vitro was also achieved. Ex vivo studies (using freshly excised porcine skin as a human analog) were also carried out to verify the antibacterial effect of the plasma jet treatment at this optimal operational frequency and to investigate the effect of treatment duration on the reduction of bacterial load. The plasma jet treatment was found to yield a 4 log reduction in bacterial load after 6 min of treatment, with no observable adverse effects on the treatment surface. The gram-negative bacterial cell lines were found to be far more susceptible to the atmospheric plasma treatments than the gram-positive bacteria. Flow cytometric analysis of plasma treated bacterial cells (Escherichia coli) was conducted in order to attain a fundamental understanding of the mode of action of the treatment on bacteria at a cellular level. This study showed that after treatment with the plasma jet, E. coli cells progressed through the following steps of cell death; the inactivation of transport systems, followed by depolarization of the cytoplasmic membrane, and finally permeabilization of the cell wall.

  1. Development of method for evaluating cell hardness and correlation between bacterial spore hardness and durability

    Science.gov (United States)

    2012-01-01

    Background Despite the availability of conventional devices for making single-cell manipulations, determining the hardness of a single cell remains difficult. Here, we consider the cell to be a linear elastic body and apply Young’s modulus (modulus of elasticity), which is defined as the ratio of the repulsive force (stress) in response to the applied strain. In this new method, a scanning probe microscope (SPM) is operated with a cantilever in the “contact-and-push” mode, and the cantilever is applied to the cell surface over a set distance (applied strain). Results We determined the hardness of the following bacterial cells: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and five Bacillus spp. In log phase, these strains had a similar Young’s modulus, but Bacillus spp. spores were significantly harder than the corresponding vegetative cells. There was a positive, linear correlation between the hardness of bacterial spores and heat or ultraviolet (UV) resistance. Conclusions Using this technique, the hardness of a single vegetative bacterial cell or spore could be determined based on Young’s modulus. As an application of this technique, we demonstrated that the hardness of individual bacterial spores was directly proportional to heat and UV resistance, which are the conventional measures of physical durability. This technique allows the rapid and direct determination of spore durability and provides a valuable and innovative method for the evaluation of physical properties in the field of microbiology. PMID:22676476

  2. Development of method for evaluating cell hardness and correlation between bacterial spore hardness and durability

    Directory of Open Access Journals (Sweden)

    Nakanishi Koichi

    2012-06-01

    Full Text Available Abstract Background Despite the availability of conventional devices for making single-cell manipulations, determining the hardness of a single cell remains difficult. Here, we consider the cell to be a linear elastic body and apply Young’s modulus (modulus of elasticity, which is defined as the ratio of the repulsive force (stress in response to the applied strain. In this new method, a scanning probe microscope (SPM is operated with a cantilever in the “contact-and-push” mode, and the cantilever is applied to the cell surface over a set distance (applied strain. Results We determined the hardness of the following bacterial cells: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and five Bacillus spp. In log phase, these strains had a similar Young’s modulus, but Bacillus spp. spores were significantly harder than the corresponding vegetative cells. There was a positive, linear correlation between the hardness of bacterial spores and heat or ultraviolet (UV resistance. Conclusions Using this technique, the hardness of a single vegetative bacterial cell or spore could be determined based on Young’s modulus. As an application of this technique, we demonstrated that the hardness of individual bacterial spores was directly proportional to heat and UV resistance, which are the conventional measures of physical durability. This technique allows the rapid and direct determination of spore durability and provides a valuable and innovative method for the evaluation of physical properties in the field of microbiology.

  3. MECHANISM OF ACTION OF ANTIBIOTICS WHICH INHIBIT SYNTHESIS OF BACTERIAL CELL WALL

    Directory of Open Access Journals (Sweden)

    Indira Mujezinović

    2013-03-01

    Full Text Available Bacterial cell possess a cell wall, which is a main difference from mammalian cells. Its basic function is to provide the strength of bacteria, keeps its shape and provides an unusually high internal osmotic pressure. Synthesis of (construction of bacterial cell wall occurs in at least three phases. All of these three phases can be influence by a variety of antibiotics in way to inhibit its synthesis. The most important drugs that act in this manner are ß-lactam antibiotics (penicillins, cephalosporins, cephamycins and other ß-lactams. They interfere with the synthesis of the bacterial cell wall peptidoglycan. After attachment to penicillin binding proteins (PBP on bacteria, they inhibit the transpeptidation enzyme that cross-links the peptide chain attached to the backbone of the peptidoglycan. The final bactericidal event is the inactivation of an inhibitor of autolytic enzymes in the cell wall, wich leads to lysis of the bacteria. Vancomycin inhibits the release of the building block unit from the carrier, thus preventing its addition to the growing end of the peptidoglycan. Cycloserine, which is a structural analogue of D-alanine, prevents the addition of the two terminal alanine residue to the initial tripeptide side-chain on N-acetylmuramic acid by competitive inhibition. Bacitracin interferes with the regeneration of the lipid carrier by blocking its dephosphorylation. Key words: bacterial cell wall, paptidoglycan, antibiotics, ß-lactams

  4. Bacterial cell-cell communication in the host via RRNPP peptide-binding regulators

    Directory of Open Access Journals (Sweden)

    David ePerez-Pascual

    2016-05-01

    Full Text Available Human microbiomes are composed of complex and dense bacterial consortia. In these environments, bacteria are able to react quickly to change by coordinating their gene expression at the population level via small signaling molecules. In Gram-positive bacteria, cell-cell communication is mostly mediated by peptides that are released into the extracellular environment. Cell-cell communication based on these peptides is especially widespread in the group Firmicutes, in which they regulate a wide array of biological processes, including functions related to host-microbe interactions. Among the different agents of communication, the RRNPP family of cytoplasmic transcriptional regulators, together with their cognate re-internalized signaling peptides, represents a group of emerging importance. RRNPP members that have been studied so far are found mainly in species of bacilli, streptococci, and enterococci. These bacteria are characterized as both human commensal and pathogenic, and share different niches in the human body with other microorganisms. The goal of this mini-review is to present the current state of research on the biological relevance of RRNPP mechanisms in the context of the host, highlighting their specific roles in commensalism or virulence.

  5. Mechanisms of bacterial morphogenesis: evolutionary cell biology approaches provide new insights.

    Science.gov (United States)

    Jiang, Chao; Caccamo, Paul D; Brun, Yves V

    2015-04-01

    How Darwin's "endless forms most beautiful" have evolved remains one of the most exciting questions in biology. The significant variety of bacterial shapes is most likely due to the specific advantages they confer with respect to the diverse environments they occupy. While our understanding of the mechanisms generating relatively simple shapes has improved tremendously in the last few years, the molecular mechanisms underlying the generation of complex shapes and the evolution of shape diversity are largely unknown. The emerging field of bacterial evolutionary cell biology provides a novel strategy to answer this question in a comparative phylogenetic framework. This relatively novel approach provides hypotheses and insights into cell biological mechanisms, such as morphogenesis, and their evolution that would have been difficult to obtain by studying only model organisms. We discuss the necessary steps, challenges, and impact of integrating "evolutionary thinking" into bacterial cell biology in the genomic era. © 2015 WILEY Periodicals, Inc.

  6. Single-bacterium nanomechanics in biomedicine: unravelling the dynamics of bacterial cells

    International Nuclear Information System (INIS)

    Aguayo, S; Bozec, L; Donos, N; Spratt, D

    2015-01-01

    The use of the atomic force microscope (AFM) in microbiology has progressed significantly throughout the years since its first application as a high-resolution imaging instrument. Modern AFM setups are capable of characterizing the nanomechanical behaviour of bacterial cells at both the cellular and molecular levels, where elastic properties and adhesion forces of single bacterium cells can be examined under different experimental conditions. Considering that bacterial and biofilm-mediated infections continue to challenge the biomedical field, it is important to understand the biophysical events leading towards bacterial adhesion and colonization on both biological and non-biological substrates. The purpose of this review is to present the latest findings concerning the field of single-bacterium nanomechanics, and discuss future trends and applications of nanoindentation and single-cell force spectroscopy techniques in biomedicine. (topical review)

  7. Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells.

    Science.gov (United States)

    Furuse, Yuki; Finethy, Ryan; Saka, Hector A; Xet-Mull, Ana M; Sisk, Dana M; Smith, Kristen L Jurcic; Lee, Sunhee; Coers, Jörn; Valdivia, Raphael H; Tobin, David M; Cullen, Bryan R

    2014-01-01

    MicroRNAs are expressed by all multicellular organisms and play a critical role as post-transcriptional regulators of gene expression. Moreover, different microRNA species are known to influence the progression of a range of different diseases, including cancer and microbial infections. A number of different human viruses also encode microRNAs that can attenuate cellular innate immune responses and promote viral replication, and a fungal pathogen that infects plants has recently been shown to express microRNAs in infected cells that repress host cell immune responses and promote fungal pathogenesis. Here, we have used deep sequencing of total expressed small RNAs, as well as small RNAs associated with the cellular RNA-induced silencing complex RISC, to search for microRNAs that are potentially expressed by intracellular bacterial pathogens and translocated into infected animal cells. In the case of Legionella and Chlamydia and the two mycobacterial species M. smegmatis and M. tuberculosis, we failed to detect any bacterial small RNAs that had the characteristics expected for authentic microRNAs, although large numbers of small RNAs of bacterial origin could be recovered. However, a third mycobacterial species, M. marinum, did express an ∼ 23-nt small RNA that was bound by RISC and derived from an RNA stem-loop with the characteristics expected for a pre-microRNA. While intracellular expression of this candidate bacterial microRNA was too low to effectively repress target mRNA species in infected cultured cells in vitro, artificial overexpression of this potential bacterial pre-microRNA did result in the efficient repression of a target mRNA. This bacterial small RNA therefore represents the first candidate microRNA of bacterial origin.

  8. Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells.

    Directory of Open Access Journals (Sweden)

    Yuki Furuse

    Full Text Available MicroRNAs are expressed by all multicellular organisms and play a critical role as post-transcriptional regulators of gene expression. Moreover, different microRNA species are known to influence the progression of a range of different diseases, including cancer and microbial infections. A number of different human viruses also encode microRNAs that can attenuate cellular innate immune responses and promote viral replication, and a fungal pathogen that infects plants has recently been shown to express microRNAs in infected cells that repress host cell immune responses and promote fungal pathogenesis. Here, we have used deep sequencing of total expressed small RNAs, as well as small RNAs associated with the cellular RNA-induced silencing complex RISC, to search for microRNAs that are potentially expressed by intracellular bacterial pathogens and translocated into infected animal cells. In the case of Legionella and Chlamydia and the two mycobacterial species M. smegmatis and M. tuberculosis, we failed to detect any bacterial small RNAs that had the characteristics expected for authentic microRNAs, although large numbers of small RNAs of bacterial origin could be recovered. However, a third mycobacterial species, M. marinum, did express an ∼ 23-nt small RNA that was bound by RISC and derived from an RNA stem-loop with the characteristics expected for a pre-microRNA. While intracellular expression of this candidate bacterial microRNA was too low to effectively repress target mRNA species in infected cultured cells in vitro, artificial overexpression of this potential bacterial pre-microRNA did result in the efficient repression of a target mRNA. This bacterial small RNA therefore represents the first candidate microRNA of bacterial origin.

  9. [Sizes of bacterial cells in soils determined by cascade filtration technique].

    Science.gov (United States)

    Polianskaia, L M; Gorodnichev, R B; Zviagintsev, D G

    2013-01-01

    This paper studies the number of bacteria in typical chernozem and mountain-meadow soil by the traditional method and the cascade filtration technique. The total number of bacteria in these soils, which was obtained in filters of different diameters during filtering the suspension of a certain amount, is 1.5-5 times higher than that obtained by the traditional method. In the structure of the bacterial biomass in both soils, the biomass of bacterial cells with a diameter of 0.38-0.43 microm was dominating by 8-90%. In the typical chernozem, the biomass of cells with a diameter of 0.17 microm was slightly more than 1%; in the mountain-meadow soil, the percentage of the biomass of cells with a diameter of 0.17 microm increased by 5%. The average volume and diameter of the bacteria in the studied soils were calculated. In typical chernozem, the average volume of bacterial cells was equal to 0.0046 microm3 and the diameter was 0.206 microm. In the mountain-meadow soils, these values were slightly lower, 0.0038 microm3 and 0.194 microm, respectively. The biomass of the bacterial cells, which is usually calculated based on the cell volume of 0.1 microm3, is overestimated by about five times when counting the number on the filters. The percentage of the real biomass of soil bacteria is traditionally much lower than that estimated.

  10. Cell motility and antibiotic tolerance of bacterial swarms

    Science.gov (United States)

    Zuo, Wenlong

    Many bacteria species can move across moist surfaces in a coordinated manner known as swarming. It is reported that swarm cells show higher tolerance to a wide variety of antibiotics than planktonic cells. We used the model bacterium E. coli to study how motility affects the antibiotic tolerance of swarm cells. Our results provide new insights for the control of pathogenic invasion via regulating cell motility. Mailing address: Room 306 Science Centre North Block, The Chinese University of Hong Kong, Shatin, N.T. Hong Kong SAR. Phone: +852-3943-6354. Fax: +852-2603-5204. E-mail: zwlong@live.com.

  11. Subversion of the B-cell compartment during parasitic, bacterial, and viral infections

    OpenAIRE

    Borhis, Gwenoline; Richard, Yolande

    2015-01-01

    International audience; AbstractRecent studies on HIV infection have identified new human B-cell subsets with a potentially important impact on anti-viral immunity. Current work highlights the occurrence of similar B-cell alterations in other viral, bacterial, and parasitic infections, suggesting that common strategies have been developed by pathogens to counteract protective immunity. For this review, we have selected key examples of human infections for which B-cell alterations have been de...

  12. Cooperation between Monocyte-Derived Cells and Lymphoid Cells in the Acute Response to a Bacterial Lung Pathogen.

    Directory of Open Access Journals (Sweden)

    Andrew S Brown

    2016-06-01

    Full Text Available Legionella pneumophila is the causative agent of Legionnaires' disease, a potentially fatal lung infection. Alveolar macrophages support intracellular replication of L. pneumophila, however the contributions of other immune cell types to bacterial killing during infection are unclear. Here, we used recently described methods to characterise the major inflammatory cells in lung after acute respiratory infection of mice with L. pneumophila. We observed that the numbers of alveolar macrophages rapidly decreased after infection coincident with a rapid infiltration of the lung by monocyte-derived cells (MC, which, together with neutrophils, became the dominant inflammatory cells associated with the bacteria. Using mice in which the ability of MC to infiltrate tissues is impaired it was found that MC were required for bacterial clearance and were the major source of IL12. IL12 was needed to induce IFNγ production by lymphoid cells including NK cells, memory T cells, NKT cells and γδ T cells. Memory T cells that produced IFNγ appeared to be circulating effector/memory T cells that infiltrated the lung after infection. IFNγ production by memory T cells was stimulated in an antigen-independent fashion and could effectively clear bacteria from the lung indicating that memory T cells are an important contributor to innate bacterial defence. We also determined that a major function of IFNγ was to stimulate bactericidal activity of MC. On the other hand, neutrophils did not require IFNγ to kill bacteria and alveolar macrophages remained poorly bactericidal even in the presence of IFNγ. This work has revealed a cooperative innate immune circuit between lymphoid cells and MC that combats acute L. pneumophila infection and defines a specific role for IFNγ in anti-bacterial immunity.

  13. Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency.

    Science.gov (United States)

    Tu, Qiang; Yin, Jia; Fu, Jun; Herrmann, Jennifer; Li, Yuezhong; Yin, Yulong; Stewart, A Francis; Müller, Rolf; Zhang, Youming

    2016-04-20

    Bacterial competent cells are essential for cloning, construction of DNA libraries, and mutagenesis in every molecular biology laboratory. Among various transformation methods, electroporation is found to own the best transformation efficiency. Previous electroporation methods are based on washing and electroporating the bacterial cells in ice-cold condition that make them fragile and prone to death. Here we present simple temperature shift based methods that improve DNA transformation and recombineering efficiency in E. coli and several other gram-negative bacteria thereby economizing time and cost. Increased transformation efficiency of large DNA molecules is a significant advantage that might facilitate the cloning of large fragments from genomic DNA preparations and metagenomics samples.

  14. The interaction of bacterial magnetosomes and human liver cancer cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Pingping, E-mail: wangpp@mail.iee.ac.cn [Beijing Key Laboratory of Bioelectromagnetism, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190 (China); Chen, Chuanfang [Beijing Key Laboratory of Bioelectromagnetism, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190 (China); Chen, Changyou [Beijing Key Laboratory of Bioelectromagnetism, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Yue; Pan, Weidong; Song, Tao [Beijing Key Laboratory of Bioelectromagnetism, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190 (China)

    2017-04-01

    As the biogenic magnetic nanomaterial, bacterial magnetic nanoparticles, namely magnetosomes, provide many advantages for potential biomedical applications. As such, interactions among magnetosomes and target cells should be elucidated to develop their bioapplications and evaluate their biocompatibilities. In this study, the interaction of magnetosomes and human liver cancer HepG2 cells was examined. Prussian blue staining revealed numerous stained particles in or on the cells. Intracellular iron concentrations, measured through inductively coupled plasma optical emission spectroscopy, increased with the increasing concentration of the magnetosomes. Transmission electron microscopy images showed that magnetosomes could be internalized in cells, mainly encapsulated in membrane vesicles, such as endosomes and lysosomes, and partly found as free particles in the cytosol. Some of the magnetosomes on cellular surfaces were encapsulated through cell membrane ruffling, which is the initiating process of endocytosis. Applying low temperature treatment and using specific endocytic inhibitors, we validated that macropinocytosis and clathrin-mediated endocytosis were involved in magnetosome uptake by HepG2 cells. Consequently, we revealed the interaction and intrinsic endocytic mechanisms of magnetosomes and HepG2 cells. This study provides a basis for the further research on bacterial magnetosome applications in liver diseases. - Highlights: • Bacterial magnetosomes interact with HepG2 cells in a dose-dependent manner. • Magnetosomes are wrapped by membrane ruffling on cell surface. • Internalized magnetosomes mainly localize in endosomes and lysosomes. • Macropinocytosis and CME are involved in the cellular uptake of magnetosomes.

  15. SuperSegger: robust image segmentation, analysis and lineage tracking of bacterial cells.

    Science.gov (United States)

    Stylianidou, Stella; Brennan, Connor; Nissen, Silas B; Kuwada, Nathan J; Wiggins, Paul A

    2016-11-01

    Many quantitative cell biology questions require fast yet reliable automated image segmentation to identify and link cells from frame-to-frame, and characterize the cell morphology and fluorescence. We present SuperSegger, an automated MATLAB-based image processing package well-suited to quantitative analysis of high-throughput live-cell fluorescence microscopy of bacterial cells. SuperSegger incorporates machine-learning algorithms to optimize cellular boundaries and automated error resolution to reliably link cells from frame-to-frame. Unlike existing packages, it can reliably segment microcolonies with many cells, facilitating the analysis of cell-cycle dynamics in bacteria as well as cell-contact mediated phenomena. This package has a range of built-in capabilities for characterizing bacterial cells, including the identification of cell division events, mother, daughter and neighbouring cells, and computing statistics on cellular fluorescence, the location and intensity of fluorescent foci. SuperSegger provides a variety of postprocessing data visualization tools for single cell and population level analysis, such as histograms, kymographs, frame mosaics, movies and consensus images. Finally, we demonstrate the power of the package by analyzing lag phase growth with single cell resolution. © 2016 John Wiley & Sons Ltd.

  16. Contribution of bacterial cell nitrogen to soil humic fractions

    International Nuclear Information System (INIS)

    Knowles, R.; Barro, L.

    1981-01-01

    Living cells of Serratia marcescens, uniformly labelled with 15 N, were added to samples of maple (Acer saccharum) and black spruce (Picea mariana) forest soils. After different periods of incubation from zero time to 100 days, the soils were subjected to alkali-acid and phenol extraction to provide humic acid, fulvic acid, humin and 'humoprotein' fractions. Significant amounts of the cell nitrogen were recovered in the humic and fulvic acids immediately after addition. After incubation, less cell nitrogen appeared in the humic acid and more in the fulvic acid. The amount of cell nitrogen recovered in the humin fraction increased with incubation. Roughly 5 to 10 per cent of the added cell nitrogen was found as amino acid nitrogen from humoprotein in a phenol extract of the humic acid. The data are consistent with the occurrence of co-precipitation of biologically labile biomass nitrogen compounds with humic polymers during the alkaline extraction procedure involved in the humic-fulvic fractionation. (orig.)

  17. Flow cytometric bacterial cell counts challenge conventional heterotrophic plate counts for routine microbiological drinking water monitoring.

    Science.gov (United States)

    Van Nevel, S; Koetzsch, S; Proctor, C R; Besmer, M D; Prest, E I; Vrouwenvelder, J S; Knezev, A; Boon, N; Hammes, F

    2017-04-15

    Drinking water utilities and researchers continue to rely on the century-old heterotrophic plate counts (HPC) method for routine assessment of general microbiological water quality. Bacterial cell counting with flow cytometry (FCM) is one of a number of alternative methods that challenge this status quo and provide an opportunity for improved water quality monitoring. After more than a decade of application in drinking water research, FCM methodology is optimised and established for routine application, supported by a considerable amount of data from multiple full-scale studies. Bacterial cell concentrations obtained by FCM enable quantification of the entire bacterial community instead of the minute fraction of cultivable bacteria detected with HPC (typically water samples per day, depending on the laboratory and selected staining procedure(s). Moreover, many studies have shown FCM total (TCC) and intact (ICC) cell concentrations to be reliable and robust process variables, responsive to changes in the bacterial abundance and relevant for characterising and monitoring drinking water treatment and distribution systems. The purpose of this critical review is to initiate a constructive discussion on whether FCM could replace HPC in routine water quality monitoring. We argue that FCM provides a faster, more descriptive and more representative quantification of bacterial abundance in drinking water. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Peptide Dendrons as Thermal-Stability Amplifiers for Immunoglobulin G1 Monoclonal Antibody Biotherapeutics.

    Science.gov (United States)

    Bansal, Rohit; Dhawan, Sameer; Chattopadhyay, Soumili; Maurya, Govind P; Haridas, V; Rathore, Anurag S

    2017-10-18

    Biotherapeutics such as monoclonal antibodies (mAbs) have a major share of the pharmaceutical industry for treatment of life-threatening chronic diseases such as cancer, skin ailments, and immune disorders. Instabilities such as aggregation, fragmentation, oxidation, and reduction have resulted in the practice of storing these products at low temperatures (-80 to -20 °C). However, reliable storage at these temperatures can be a challenge, particularly in developing and underdeveloped countries; hence, lately, there has been a renewed interest in creating formulations that would offer stability at higher temperatures (25 to 55 °C). Most therapeutic formulations contain excipients such as salts, sugars, amino acids, surfactants, and polymers to provide stability to the biotherapeutic, but their efficacy at high temperatures is limited. The current work proposes the use of peptide dendrons of different generations to create formulations that would be stable at high temperature. Among these dendrons, third-generation lysine dendron L6 has been identified to provide the highest stability to mAbs, as demonstrated by a host of analytical techniques such as size-exclusion chromatography (SEC), dynamic light scattering (DLS), Nanoparticle tracking Analysis (NTA), and circular dichroism (CD). The biocompatibility of these dendrons was confirmed by hemolytic activity tests. Non-interference of the dendrons with the activity of the mAb was confirmed using a surface plasmon resonance (SPR) based activity assay. We hope that this study will stimulate utilization of such higher-generation dendrons for enhancing the thermal stability of mAbs.

  19. Glycerol Monolaurate Inhibits Lipase Production by Clinical Ocular Isolates Without Affecting Bacterial Cell Viability.

    Science.gov (United States)

    Flanagan, Judith Louise; Khandekar, Neeta; Zhu, Hua; Watanabe, Keizo; Markoulli, Maria; Flanagan, John Terence; Papas, Eric

    2016-02-01

    We sought to determine the relative lipase production of a range of ocular bacterial isolates and to assess the efficacy of glycerol monolaurate (GML) in inhibiting this lipase production in high lipase-producing bacteria without affecting bacterial cell growth. Staphylococcus aureus,Staphylococcus epidermidis,Propionibacterium acnes, and Corynebacterium spp. were inoculated at a density of 10(6)/mL in varying concentrations of GML up to 25 μg/mL for 24 hours at 37 °C with constant shaking. Bacterial suspensions were centrifuged, bacterial cell density was determined, and production of bacterial lipase was quantified using a commercial lipase assay kit. Staphylococcus spp. produced high levels of lipase activity compared with P. acnes and Corynebacterium spp. GML inhibited lipase production by Staphylococcal spp. in a dose-dependent manner, with S. epidermidis lipase production consistently more sensitive to GML than S. aureus. Glycerol monolaurate showed significant (P lipase inhibition above concentrations of 15 μg/mL in S. aureus and was not cytotoxic up to 25 μg/mL. For S. epidermidis, GML showed significant (P lipase inhibition above 7.5 μg/mL. Lipase activity varied between species and between strains. Staphylococcal spp. produced higher lipase activity compared with P. acnes and Corynebacterium spp. Glycerol monolaurate inhibited lipase production by S. aureus and S. epidermidis at concentrations that did not adversely affect bacterial cell growth. GML can be used to inhibit ocular bacterial lipase production without proving detrimental to commensal bacteria viability.

  20. In-vitro analysis of APA microcapsules for oral delivery of live bacterial cells.

    Science.gov (United States)

    Chen, H; Ouyang, W; Jones, M; Haque, T; Lawuyi, B; Prakash, S

    2005-08-01

    Oral administration of microcapsules containing live bacterial cells has potential as an alternative therapy for several diseases. This article evaluates the suitability of the alginate-poly-L-lysine-alginate (APA) microcapsules for oral delivery of live bacterial cells, in-vitro, using a dynamic simulated human gastro-intestinal (GI) model. Results showed that the APA microcapsules were morphologically stable in the simulated stomach conditions, but did not retain their structural integrity after a 3-day exposure in simulated human GI media. The microbial populations of the tested bacterial cells and the activities of the tested enzymes in the simulated human GI suspension were not substantially altered by the presence of the APA microcapsules, suggesting that there were no significant adverse effects of oral administration of the APA microcapsules on the flora of the human gastrointestinal tract. When the APA microcapsules containing Lactobacillus plantarum 80 (LP80) were challenged in the simulated gastric medium (pH = 2.0), 80.0% of the encapsulated cells remained viable after a 5-min incubation; however, the viability decreased considerably (8.3%) after 15 min and dropped to 2.6% after 30 min and lower than 0.2% after 60 min, indicating the limitations of the currently obtainable APA membrane for oral delivery of live bacteria. Further in-vivo studies are required before conclusions can be made concerning the inadequacy of APA microcapsules for oral delivery of live bacterial cells.

  1. Use of cyclodextrin and its derivatives for increased transformation efficiency of competent bacterial cells.

    Science.gov (United States)

    Aachmann, Finn Lillelund; Aune, Trond Erik Vee

    2009-06-01

    Methodologies for introduction of DNA into cells are essential in molecular genetics and vital for applications such as genetic engineering and gene therapy. The use of cyclodextrins (CyDs) for increased efficiency of introducing DNA into eukaryotic cells (transfection) has been reported, but CyDs' effect on the introduction of DNA into bacterial cells (transformation) is unknown. Here, we have investigated the potential of using CyDs in the transformation of chemically competent in-house, commercially available, and, on non-competent bacterial cells, with plasmid DNA of two different sizes. Possible interactions between CyDs and DNA were studied with nuclear magnetic resonance (NMR) spectroscopy. The presence of CyDs resulted in an up to fourfold increment of the transformation rate for in-house cells, with beta-CyD and derivates giving the strongest effect. For commercial cells and transformation with megaplasmids, a more moderate effect around 1.4-fold was obtained. However, CyDs have little or no effect on DNA uptake by noncompetent cells. Results obtained from NMR spectroscopy show no interactions between CyDs and DNA-like molecules, which indicated that the CyDs' effect is related to the bacterial cell wall.

  2. Vehicles, Replicators, and Intercellular Movement of Genetic Information: Evolutionary Dissection of a Bacterial Cell

    Directory of Open Access Journals (Sweden)

    Matti Jalasvuori

    2012-01-01

    Full Text Available Prokaryotic biosphere is vastly diverse in many respects. Any given bacterial cell may harbor in different combinations viruses, plasmids, transposons, and other genetic elements along with their chromosome(s. These agents interact in complex environments in various ways causing multitude of phenotypic effects on their hosting cells. In this discussion I perform a dissection for a bacterial cell in order to simplify the diversity into components that may help approach the ocean of details in evolving microbial worlds. The cell itself is separated from all the genetic replicators that use the cell vehicle for preservation and propagation. I introduce a classification that groups different replicators according to their horizontal movement potential between cells and according to their effects on the fitness of their present host cells. The classification is used to discuss and improve the means by which we approach general evolutionary tendencies in microbial communities. Moreover, the classification is utilized as a tool to help formulating evolutionary hypotheses and to discuss emerging bacterial pathogens as well as to promote understanding on the average phenotypes of different replicators in general. It is also discussed that any given biosphere comprising prokaryotic cell vehicles and genetic replicators may naturally evolve to have horizontally moving replicators of various types.

  3. Effect of micro- and nanoscale topography on the adhesion of bacterial cells to solid surfaces.

    Science.gov (United States)

    Hsu, Lillian C; Fang, Jean; Borca-Tasciuc, Diana A; Worobo, Randy W; Moraru, Carmen I

    2013-04-01

    Attachment and biofilm formation by bacterial pathogens on surfaces in natural, industrial, and hospital settings lead to infections and illnesses and even death. Minimizing bacterial attachment to surfaces using controlled topography could reduce the spreading of pathogens and, thus, the incidence of illnesses and subsequent human and financial losses. In this context, the attachment of key microorganisms, including Escherichia coli, Listeria innocua, and Pseudomonas fluorescens, to silica and alumina surfaces with micron and nanoscale topography was investigated. The results suggest that orientation of the attached cells occurs preferentially such as to maximize their contact area with the surface. Moreover, the bacterial cells exhibited different morphologies, including different number and size of cellular appendages, depending on the topographical details of the surface to which they attached. This suggests that bacteria may utilize different mechanisms of attachment in response to surface topography. These results are important for the design of novel microbe-repellant materials.

  4. Biotherapeutic potential and mechanisms of action of colchicine.

    Science.gov (United States)

    Dubey, Kashyap Kumar; Kumar, Punit; Labrou, Nikolaos E; Shukla, Pratyoosh

    2017-12-01

    Cancer is a clinical situation caused by uncontrolled cell division and is responsible for a large number of deaths worldwide. Colchicine is a classical antimitotic, tubulin-binding agent (TBA) which is being explored for its antitumor activities, although its tubulin-binding ability leads to some toxicity toward normal cells proliferation. Colchicine derivatives are considered as potent antitumor compounds with less toxicity compared to colchicine. Derivatives with substituted functional groups at A-ring (methoxy), B-ring (acetamide) or C-ring (methoxy) have been synthesized via chemical and microbial routes and show modified bioactivities and altered tropolonic functionality. Earlier reports, in combination with our group's research findings, suggest that microbial biotransformation is an efficient choice for the production of bioactive colchicine derivatives. This route has gained significant interest in the mass production of regio-specific, cost-effective, safe and eco-friendly derivatives. The present review paper critically analyzes and discusses the development and application of colchicine derivatives as a potent antitumor molecule and their production through a microbial transformation process. The information provided in this review might assist in the stimulation of new ideas regarding the development of alternative therapeutic agent(s) for cancer treatment.

  5. The impact of metabolic state on Cd adsorption onto bacterial cells

    Science.gov (United States)

    Johnson, K.J.; Ams, D.A.; Wedel, A.N.; Szymanowski, J.E.S.; Weber, D.L.; Schneegurt, M.A.; Fein, J.B.

    2007-01-01

    This study examines the effect of bacterial metabolism on the adsorption of Cd onto Gram-positive and Gram-negative bacterial cells. Metabolically active Gram-positive cells adsorbed significantly less Cd than non-metabolizing cells. Gram-negative cells, however, showed no systematic difference in Cd adsorption between metabolizing and non-metabolizing cells. The effect of metabolism on Cd adsorption to Gram-positive cells was likely due to an influx of protons in and around the cell wall from the metabolic proton motive force, promoting competition between Cd and protons for adsorption sites on the cell wall. The relative lack of a metabolic effect on Cd adsorption onto Gram-negative compared to Gram-positive cells suggests that Cd binding in Gram-negative cells is focused in a region of the cell wall that is not reached, or is unaffected by this proton flux. Thermodynamic modeling was used to estimate that proton pumping causes the pH in the cell wall of metabolizing Gram-positive bacteria to decrease from the bulk solution value of 7.0 to approximately 5.7. ?? 2007 The Authors.

  6. Bacterial Cell Surface Adsorption of Rare Earth Elements

    Science.gov (United States)

    Jiao, Y.; Park, D.; Reed, D.; Fujita, Y.; Yung, M.; Anderko, A.; Eslamimanesh, A.

    2015-12-01

    Rare earth elements (REE) play a critical role in many emerging clean energy technologies, including high-power magnets, wind turbines, solar panels, hybrid/electric vehicle batteries and lamp phosphors. In order to sustain demand for such technologies given current domestic REE shortages, there is a need to develop new approaches for ore processing/refining and recycling of REE-containing materials. To this end, we have developed a microbially-mediated bioadsorption strategy with application towards enrichment of REE from complex mixtures. Specifically, the bacterium Caulobacter crescentus was genetically engineered to display lanthanide binding tags (LBTs), short peptides that possess high affinity and specificity for rare earth elements, on its cell surface S-layer protein. Under optimal conditions, LBT-displayed cells adsorbed greater than 5-fold more REE than control cells lacking LBTs. Competition binding experiments with a selection of REEs demonstrated that our engineered cells could facilitate separation of light- from heavy- REE. Importantly, binding of REE onto our engineered strains was much more favorable compared to non-REE metals. Finally, REE bound to the cell surface could be stripped off using citrate, providing an effective and non-toxic REE recovery method. Together, this data highlights the potential of our approach for selective REE enrichment from REE containing mixtures.

  7. In situ probing the interior of single bacterial cells at nanometer scale

    Science.gov (United States)

    Liu, Boyin; Hemayet Uddin, Md; Ng, Tuck Wah; Paterson, David L.; Velkov, Tony; Li, Jian; Fu, Jing

    2014-10-01

    We report a novel approach to probe the interior of single bacterial cells at nanometre resolution by combining focused ion beam (FIB) and atomic force microscopy (AFM). After removing layers of pre-defined thickness in the order of 100 nm on the target bacterial cells with FIB milling, AFM of different modes can be employed to probe the cellular interior under both ambient and aqueous environments. Our initial investigations focused on the surface topology induced by FIB milling and the hydration effects on AFM measurements, followed by assessment of the sample protocols. With fine-tuning of the process parameters, in situ AFM probing beneath the bacterial cell wall was achieved for the first time. We further demonstrate the proposed method by performing a spatial mapping of intracellular elasticity and chemistry of the multi-drug resistant strain Klebsiella pneumoniae cells prior to and after it was exposed to the ‘last-line’ antibiotic polymyxin B. Our results revealed increased stiffness occurring in both surface and interior regions of the treated cells, suggesting loss of integrity of the outer membrane from polymyxin treatments. In addition, the hydrophobicity measurement using a functionalized AFM tip was able to highlight the evident hydrophobic portion of the cell such as the regions containing cell membrane. We expect that the proposed FIB-AFM platform will help in gaining deeper insights of bacteria-drug interactions to develop potential strategies for combating multi-drug resistance.

  8. Bacterial glycosidases for the production of universal red blood cells

    DEFF Research Database (Denmark)

    Liu, Qiyong P; Sulzenbacher, Gerlind; Yuan, Huaiping

    2007-01-01

    Enzymatic removal of blood group ABO antigens to develop universal red blood cells (RBCs) was a pioneering vision originally proposed more than 25 years ago. Although the feasibility of this approach was demonstrated in clinical trials for group B RBCs, a major obstacle in translating this techno......Enzymatic removal of blood group ABO antigens to develop universal red blood cells (RBCs) was a pioneering vision originally proposed more than 25 years ago. Although the feasibility of this approach was demonstrated in clinical trials for group B RBCs, a major obstacle in translating...

  9. Influence of setup and carbon source on the bacterial community of biocathodes in microbial electrolysis cells

    NARCIS (Netherlands)

    Croese, Elsemiek; Jeremiasse, Adriaan W.; Marshall, Ian P.G.; Spormann, Alfred M.; Euverink, Gert-Jan W.; Geelhoed, Jeanine S.; Stams, Alfons J.M.; Plugge, Caroline M.

    2014-01-01

    The microbial electrolysis cell (MEC) biocathode has shown great potential as alternative for expensive metals as catalyst for H2synthesis. Here, the bacterial communities at the biocathode of five hydrogen producing MECs using molecular techniques were characterized. The setups differed in design

  10. Potential effect of cationic liposomes on interactions with oral bacterial cells and biofilms.

    Science.gov (United States)

    Sugano, Marika; Morisaki, Hirobumi; Negishi, Yoichi; Endo-Takahashi, Yoko; Kuwata, Hirotaka; Miyazaki, Takashi; Yamamoto, Matsuo

    2016-01-01

    Although oral infectious diseases have been attributed to bacteria, drug treatments remain ineffective because bacteria and their products exist as biofilms. Cationic liposomes have been suggested to electrostatically interact with the negative charge on the bacterial surface, thereby improving the effects of conventional drug therapies. However, the electrostatic interaction between oral bacteria and cationic liposomes has not yet been examined in detail. The aim of the present study was to examine the behavior of cationic liposomes and Streptococcus mutans in planktonic cells and biofilms. Liposomes with or without cationic lipid were prepared using a reverse-phase evaporation method. The zeta potentials of conventional liposomes (without cationic lipid) and cationic liposomes were -13 and 8 mV, respectively, and both had a mean particle size of approximately 180 nm. We first assessed the interaction between liposomes and planktonic bacterial cells with a flow cytometer. We then used a surface plasmon resonance method to examine the binding of liposomes to biofilms. We confirmed the binding behavior of liposomes with biofilms using confocal laser scanning microscopy. The interactions between cationic liposomes and S. mutans cells and biofilms were stronger than those of conventional liposomes. Microscopic observations revealed that many cationic liposomes interacted with the bacterial mass and penetrated the deep layers of biofilms. In this study, we demonstrated that cationic liposomes had higher affinity not only to oral bacterial cells, but also biofilms than conventional liposomes. This electrostatic interaction may be useful as a potential drug delivery system to biofilms.

  11. Cell-selective labeling of bacterial proteomes with an orthogonal phenylalanine amino acid reporter.

    Science.gov (United States)

    Grammel, Markus; Dossa, Paul D; Taylor-Salmon, Emma; Hang, Howard C

    2012-02-01

    Orthogonal amino acid reporters allow the selective labeling of different cell types in heterogeneous populations through the expression of engineered aminoacyl tRNA synthetases. Here, we demonstrate that para-ethynylphenylalanine (PEP) can be used as an orthogonal amino acid reporter for efficient selective labeling of an intracellular bacterial pathogen during infection. This journal is © The Royal Society of Chemistry 2012

  12. Magic bullets to fight resistance : Uncovering how peptide-antibiotics break down the bacterial cell envelope

    NARCIS (Netherlands)

    Medeiros-Silva, J.|info:eu-repo/dai/nl/288254600; Jekhmane, S.|info:eu-repo/dai/nl/412782715; Breukink, E.|info:eu-repo/dai/nl/120305100; Weingarth, M.|info:eu-repo/dai/nl/330985655

    The rapid rise of resistant bacteria urgently calls for novel antibiotics that are robust to resistance development. Ideal templates could be peptide-antibiotics that destroy the bacterial cell wall by binding to its membrane-anchored precursor lipid II at irreplaceable phosphate groups. Indeed,

  13. Bacterial meningitis in hematopoietic stem cell transplant recipients: a population-based prospective study

    NARCIS (Netherlands)

    van Veen, K. E. B.; Brouwer, M. C.; van der Ende, A.; van de Beek, D.

    2016-01-01

    We performed a nationwide prospective cohort study on the epidemiology and clinical features of community-acquired bacterial meningitis. Patients with a medical history of autologous or allogeneic hematopoietic stem cell transplantation (HSCT) were identified from the cohort performed from March

  14. Bacterial cell wall preservation during organic matter diagenesis in sediments off Peru

    DEFF Research Database (Denmark)

    Lomstein, Bente Aagaard; Niggemann, Jutta; Jørgensen, Bo Barker

    BACTERIAL CELL WALL PRESERVATION DURING ORGANIC MATTER DIAGENESIS IN SEDIMENTS OFF PERU The spatial distribution of total hydrolysable amino acids, total hydrolysable amino sugars and amino acid enantiomers (D- and L-forms) were investigated in surface sediments at 20 stations in the Peru margin: 9...

  15. The antimicrobial polymer PHMB enters cells and selectively condenses bacterial chromosomes

    DEFF Research Database (Denmark)

    Chindera, Kantaraja; Mahato, Manohar; Sharma, Ashwani Kumar

    2016-01-01

    mixed with isolated bacterial chromosomal DNA and its effects on growth were suppressed by pairwise combination with the DNA binding ligand Hoechst 33258. PHMB also entered mammalian cells, but was trapped within endosomes and excluded from nuclei. Therefore, PHMB displays differential access...

  16. Increased electrical output when a bacterial ABTS oxidizer is used in a microbial fuel cell

    Science.gov (United States)

    Microbial fuel cells (MFCs) are a technology that provides electrical energy from the microbial oxidation of organic compounds. Most MFCs use oxygen as the oxidant in the cathode chamber. The present study examined the formation in culture of an unidentified bacterial oxidant and investigated the ...

  17. Different binarization processes validated against manual counts of fluorescent bacterial cells

    NARCIS (Netherlands)

    Tamminga, Gerrit G.; Paulitsch-Fuchs, Astrid H.; Jansen, Gijsbert J.; Euverink, Gert-Jan W.

    State of the art software methods (such as fixed value approaches or statistical approaches) to create a binary image of fluorescent bacterial cells are not as accurate and precise as they should be for counting bacteria and measuring their area. To overcome these bottlenecks, we introduce

  18. Influence of setup and carbon source on the bacterial community of biocathodes in microbial electrolysis cells

    NARCIS (Netherlands)

    Croesea, E.; Jeremiasse, A.W.; Marshall, I.P.G.; Spormann, A.M.; Euverink, G.J.W.; Geelhoed, J.S.; Stams, A.J.M.; Plugge, C.M.

    2014-01-01

    The microbial electrolysis cell (MEC) biocathode has shown great potential as alternative for expensive metals as catalyst for H2 synthesis. Here, the bacterial communities at the biocathode of five hydrogen producing MECs using molecular techniques were characterized. The setups differed in design

  19. Programmable bacterial catalysis – designing cells for biosynthesis of value-added compounds

    NARCIS (Netherlands)

    Lam, M.C.; Suarez Diez, M.; Godinho, M.; Martins Dos Santos, V.A.P.

    2012-01-01

    Bacteria have long been used for the synthesis of a wide range of useful proteins and compounds. The developments of new bioprocesses and improvements of existing strategies for syntheses of valuable products in various bacterial cell hosts have their own challenges and limitations. The field of

  20. Soluble triggering receptor expressed on myeloid cells 1: a biomarker for bacterial meningitis

    NARCIS (Netherlands)

    Determann, Rogier M.; Weisfelt, Martijn; de Gans, Jan; van der Ende, Arie; Schultz, Marcus J.; van de Beek, Diederik

    2006-01-01

    OBJECTIVE: To evaluate whether soluble triggering receptor expressed on myeloid cells 1 (sTREM-1) in CSF can serve as a biomarker for the presence of bacterial meningitis and outcome in patients with this disease. DESIGN: Retrospective study of diagnostic accuracy. SETTING AND PATIENTS: CSF was

  1. Life without a cell membrane: Challenging the specificity of bacterial endophytes within Bryopsis (Bryopsidales, Chlorophyta

    Directory of Open Access Journals (Sweden)

    Hollants Joke

    2011-11-01

    Full Text Available Abstract Background The siphonous green macroalga Bryopsis has some remarkable characteristics. Besides hosting a rich endophytic bacterial flora, Bryopsis also displays extraordinary wound repair and propagation mechanisms. This latter feature includes the formation of protoplasts which can survive in the absence of a cell membrane for several minutes before regenerating into new individuals. This transient 'life without a membrane' state, however, challenges the specificity of the endophytic bacterial communities present and raises the question whether these bacteria are generalists, which are repeatedly acquired from the environment, or if there is some specificity towards the Bryopsis host. Results To answer this question, we examined the temporal stability and the uniqueness of endobiotic bacterial communities within Bryopsis samples from the Mexican west coast after prolonged cultivation. DGGE analysis revealed that Bryopsis endophytic bacterial communities are rather stable and clearly distinct from the epiphytic and surrounding cultivation water bacterial communities. Although these endogenous communities consist of both facultative and obligate bacteria, results suggest that Bryopsis owns some intrinsic mechanisms to selectively maintain and/or attract specific bacteria after repeated wounding events in culture. Conclusions This suggests that Bryopsis algae seem to master transient stages of life without a cell membrane well as they harbor specific - and possibly ecological significant - endophytic bacteria.

  2. The Disruptive Effect of Lysozyme on the Bacterial Cell Wall Explored by an "In-Silico" Structural Outlook

    Science.gov (United States)

    Primo, Emiliano D.; Otero, Lisandro H.; Ruiz, Francisco; Klinke, Sebastián; Giordano, Walter

    2018-01-01

    The bacterial cell wall, a structural unit of peptidoglycan polymer comprised of glycan strands consisting of a repeating disaccharide motif [N-acetylglucosamine (NAG) and N-acetylmuramylpentapeptide (NAM pentapeptide)], encases bacteria and provides structural integrity and protection. Lysozymes are enzymes that break down the bacterial cell wall…

  3. Mass Cytometry for Detection of Silver at the Bacterial Single Cell Level

    Directory of Open Access Journals (Sweden)

    Yuting Guo

    2017-07-01

    Full Text Available Background: Mass cytometry (Cytometry by Time of Flight, CyTOF allows single-cell characterization on the basis of specific metal-based cell markers. In addition, other metals in the mass range such as silver can be detected per cell. Bacteria are known to be sensible to silver and a protocol was developed to measure both the number of affected cells per population and the quantities of silver per cell.Methods: For mass cytometry ruthenium red was used as a marker for all cells of a population while parallel application of cisplatin discriminated live from dead cells. Silver quantities per cell and frequencies of silver containing cells in a population were measured by mass cytometry. In addition, live/dead subpopulations were analyzed by flow cytometry and distinguished by cell sorting based on ruthenium red and propidium iodide double staining. Verification of the cells’ silver load was performed on the bulk level by using ICP-MS in combination with cell sorting. The protocol was developed by conveying both, fast and non-growing Pseudomonas putida cells as test organisms.Results: A workflow for labeling bacteria in order to be analyzed by mass cytometry was developed. Three different parameters were tested: ruthenium red provided counts for all bacterial cells in a population while consecutively applied cisplatin marked the frequency of dead cells. Apparent population heterogeneity was detected by different frequencies of silver containing cells. Silver quantities per cell were also well measurable. Generally, AgNP-10 treatment caused higher frequencies of dead cells, higher frequencies of silver containing cells and higher per-cell silver quantities. Due to an assumed chemical equilibrium of free and bound silver ions live and dead cells were associated with silver in equal quantities and this preferably during exponential growth. With ICP-MS up to 1.5 fg silver per bacterial cell were detected.Conclusion: An effective mass cytometry

  4. Bacterial conjugation in the cytoplasm of mouse cells.

    NARCIS (Netherlands)

    Lim, Y.M.; Groof, A.J.C. de; Bhattacharjee, M.K.; Figurski, D.H.; Schon, E.A.

    2008-01-01

    Intracellular pathogenic organisms such as salmonellae and shigellae are able to evade the effects of many antibiotics because the drugs are not able to penetrate the plasma membrane. In addition, these bacteria may be able to transfer genes within cells while protected from the action of drugs. The

  5. Probing the bacterial cell wall with chemical biology tools

    NARCIS (Netherlands)

    Sminia, Tjerk J.

    2017-01-01

    After DNA and proteins, carbohydrates are the third language of life. Chapter 1 introduces the reader to this class of biomolecules, also called sugars or glycans, that can be found on the outer surface of almost all cells and plays a critical role as the social messengers of a

  6. Probing the bacterial cell wall with chemical biology tools

    NARCIS (Netherlands)

    Sminia, Tjerk J.

    2017-01-01

    After DNA and proteins, carbohydrates are the third language of life. Chapter 1 introduces the reader to this class of biomolecules, also called sugars or glycans, that can be found on the outer surface of almost all cells and plays a critical role as the social messengers of a

  7. Super-resolution microscopy of living bacterial cells

    Science.gov (United States)

    Ponomareva, E. V.; Vishnyakov, I. E.; Morozova, N. E.; Polinovskaya, V. S.; Khodorkovskii, M. A.; Vedyaykin, A. D.

    2017-11-01

    Currently several methods of super-resolution optical microscopy are known. One of them – super-resolution radial fluctuations (SRRF) microscopy – was successfully used in present work to study the structures, formed by FtsZ protein in living E.coli cells with a resolution well below the diffraction limit.

  8. A novel mechanism of bacterial toxin transfer within host blood cell-derived microvesicles.

    Directory of Open Access Journals (Sweden)

    Anne-lie Ståhl

    2015-02-01

    Full Text Available Shiga toxin (Stx is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS, associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.

  9. Electron microscopy study of antioxidant interaction with bacterial cells

    Science.gov (United States)

    Plotnikov, Oleg P.; Novikova, Olga V.; Konnov, Nikolai P.; Korsukov, Vladimir N.; Gunkin, Ivan F.; Volkov, Uryi P.

    2000-10-01

    To maintain native microorganisms genotype and phenotype features a lyophylization technique is widely used. However in this case cells are affected by influences of vacuum and low temperature that cause a part of the cells population to be destruction. Another factor reduced microorganisms vitality is formation of reactive oxygen forms that damage certain biological targets (such as DNA, membranes etc.) Recently to raise microorganism's resistance against adverse condition natural and synthetic antioxidants are used. Antioxidant- are antagonists of free radicals. Introduction of antioxidants in protective medium for lyophylization increase bacteria storage life about 2,0-4,8 fold in comparison with reference samples. In the article the main results of our investigation of antioxidants interaction with microorganism cells is described. As bacteria cells we use vaccine strain yersinia pestis EV, that were grown for 48 h at 28 degree(s)C on the Hottinger agar (pH 7,2). Antioxidants are inserted on the agar surface in specimen under test. To investigate a localization of antioxidants for electron microscopy investigation, thallium organic antioxidants were used. The thallium organic compounds have an antioxidant features if thallium is in low concentration (about 1(mu) g/ml). The localization of the thallium organic antioxidants on bacteria Y. pestis EV is visible in electron microscopy images, thallium being heavy metal with high electron density. The negatively stained bacteria and bacteria thin sections with thallium organic compounds were investigated by means of transmission electron microscopy. The localization of the thallium organic compounds is clearly visible in electron micrographs as small dark spots with size about 10-80nm. Probably mechanisms of interaction of antioxidants with bacteria cells are discussed.

  10. Glyco-engineering of biotherapeutic proteins in plants.

    Science.gov (United States)

    Ko, Kisung; Ahn, Mi-Hyun; Song, Mira; Choo, Young-Kug; Kim, Hyun Soon; Ko, Kinarm; Joung, Hyouk

    2008-06-30

    Many therapeutic glycoproteins have been successfully generated in plants. Plants have advantages regarding practical and economic concerns, and safety of protein production over other existing systems. However, plants are not ideal expression systems for the production of biopharmaceutical proteins, due to the fact that they are incapable of the authentic human N-glycosylation process. The majority of therapeutic proteins are glycoproteins which harbor N-glycans, which are often essential for their stability, folding, and biological activity. Thus, several glyco-engineering strategies have emerged for the tailor-making of N-glycosylation in plants, including glycoprotein subcellular targeting, the inhibition of plant specific glycosyltranferases, or the addition of human specific glycosyltransferases. This article focuses on plant N-glycosylation structure, glycosylation variation in plant cell, plant expression system of glycoproteins, and impact of glycosylation on immunological function. Furthermore, plant glyco-engineering techniques currently being developed to overcome the limitations of plant expression systems in the production of therapeutic glycoproteins will be discussed in this review.

  11. Bacterial toxin-antitoxin gene system as containment control in yeast cells

    DEFF Research Database (Denmark)

    Kristoffersen, P.; Jensen, G. B.; Gerdes, K.

    2000-01-01

    The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae, Expression of the relE gene was highly toxic to yeast cells. However, expression...... fermentation processes in which the escape of genetically modified cells would be considered highly risky....

  12. Biomechanical Loading Modulates Proinflammatory and Bone Resorptive Mediators in Bacterial-Stimulated PDL Cells

    OpenAIRE

    Nogueira, Andressa Vilas Boas; Nokhbehsaim, Marjan; Eick, Sigrun; Bourauel, Christoph; Jäger, Andreas; Jepsen, Søren; Rossa, Carlos; Deschner, James; Cirelli, Joni Augusto

    2014-01-01

    The present study aimed to evaluate in vitro whether biomechanical loading modulates proinflammatory and bone remodeling mediators production by periodontal ligament (PDL) cells in the presence of bacterial challenge. Cells were seeded on BioFlex culture plates and exposed to Fusobacterium nucleatum ATCC 25586 and/or cyclic tensile strain (CTS) of low (CTSL) and high (CTSH) magnitudes for 1 and 3 days. Synthesis of cyclooxygenase-2 (COX2) and prostaglandin E2 (PGE2) was evaluated by ELISA. Ge...

  13. The hit principle and the mutagenic effect of ionizing radiations of different quality on bacterial cells

    International Nuclear Information System (INIS)

    Krasavin, E.A.; Kozubek, S.; Amirtaev, K.G.; Tokarova, B.

    1988-01-01

    The role of the most important methodological principle - the hit principle, worked out by N.V. Timofeeff-Ressovsky, in recent understanding of the mutagenic effect of ionizing radiation of different quality on bacterial cells has been discussed. Experimentaol results are presented which allow that mutagenic effect of ionizing radiation is determined by the influence of factors of both physical nature (the parameters of radiation and the geometry of a target) and biological nature (repair systems in cells)

  14. Procalcitonin as a biomarker of bacterial infection in sickle cell vaso-occlusive crisis.

    Science.gov (United States)

    Patel, Dilip Kumar; Mohapatra, Manoj Kumar; Thomas, Ancil George; Patel, Siris; Purohit, Prasanta

    2014-01-01

    Sickle cell anaemia (SCA) patients with vaso-occlusive crisis (VOC) have signs of inflammation and it is often difficult to diagnose a bacterial infection in them. This study was undertaken to evaluate the role of serum procalcitonin (PCT) as a biomarker of bacterial infection in acute sickle cell vaso-occlusive crisis. Hundred homozygous SCA patients were studied at Sickle Cell Clinic and Molecular Biology Laboratory, V.S.S. Medical College, Burla, Odisha, India. All the patients were divided into three categories namely category-A (VOC/ACS with SIRS but without evidence of bacterial infection - 66 patients), category-B (VOC/ACS with SIRS and either proven or suspected bacterial infection - 24 patients) and category-C (SCA patients in steady state without VOC/ACS or SIRS - 10 patients). Complete blood count, C-reactive protein (CRP) estimation and PCT measurement were done in all the patients. There was no significant difference in TLC and CRP values between category-A and B. In category-A, the PCT level was value >0.5 ng/mL with 87.5% of patients having >2 ng/mL. In category-C, PCT value was value (100%) for bacterial infection at a cutoff value of 0.5 ng/mL; whereas the specificity is excellent at a cut-off value of 2 ng/mL. SCA patients with VOC/ACS and SIRS having a PCT level of value of >2 ng/mL is indicative of bacterial infection necessitating early antimicrobial therapy.

  15. Bacterial Signaling Nucleotides Inhibit Yeast Cell Growth by Impacting Mitochondrial and Other Specifically Eukaryotic Functions.

    Science.gov (United States)

    Hesketh, Andy; Vergnano, Marta; Wan, Chris; Oliver, Stephen G

    2017-07-25

    We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP), cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA) and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR) inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection. IMPORTANCE During infections, pathogenic bacteria can release nucleotides into the cells of their eukaryotic hosts. These nucleotides are recognized as signals that contribute to the initiation of defensive immune responses that help the infected

  16. Mutagenic effect of accelerated heavy ions on bacterial cells

    Science.gov (United States)

    Boreyko, A. V.; Krasavin, E. A.

    2011-11-01

    The heavy ion accelerators of the Joint Institute for Nuclear Research were used to study the regularities and mechanisms of formation of different types of mutations in prokaryote cells. The induction of direct (lac-, ton B-, col B) mutations for Esherichia coli cells and reverse his- → His+ mutations of Salmonella typhimurium, Bacillus subtilis cells under the action of radiation in a wide range of linear energy transfer (LET) was studied. The regularities of formation of gene and structural (tonB trp-) mutations for Esherichia coli bacteria under the action of accelerated heavy ions were studied. It was demonstrated that the rate of gene mutations as a function of the dose under the action of Γ rays and accelerated heavy ions is described by linear-quadratic functions. For structural mutations, linear "dose-effect" dependences are typical. The quadratic character of mutagenesis dose curves is determined by the "interaction" of two independent "hitting" events in the course of SOS repair of genetic structures. The conclusion made was that gene mutations under the action of accelerated heavy ions are induced by δ electron regions of charged particle tracks. The methods of SOS chromotest, SOS lux test, and λ prophage induction were used to study the regularities of SOS response of cells under the action of radiations in a wide LET range. The following proposition was substantiated: the molecular basis for formation of gene mutations are cluster single-strand DNA breaks, and that for structural mutations, double-strand DNA breaks. It was found out that the LET dependence of the relative biological efficiency of accelerated ions is described by curves with a local maximum. It was demonstrated that the biological efficiency of ionizing radiations with different physical characteristics on cells with different genotype, estimated by the lethal action, induction of gene and deletion mutations, precision excision of transposons, is determined by the specific

  17. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

    Directory of Open Access Journals (Sweden)

    Chew Chieng Yeo

    2016-02-01

    Full Text Available Toxin-antitoxin (TA systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  18. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications.

    Science.gov (United States)

    Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

    2016-02-19

    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  19. Cholesterol-lowering probiotics as potential biotherapeutics for metabolic diseases.

    Science.gov (United States)

    Kumar, Manoj; Nagpal, Ravinder; Kumar, Rajesh; Hemalatha, R; Verma, Vinod; Kumar, Ashok; Chakraborty, Chaitali; Singh, Birbal; Marotta, Francesco; Jain, Shalini; Yadav, Hariom

    2012-01-01

    Cardiovascular diseases are one of the major causes of deaths in adults in the western world. Elevated levels of certain blood lipids have been reported to be the principal cause of cardiovascular disease and other disabilities in developed countries. Several animal and clinical trials have shown a positive association between cholesterol levels and the risks of coronary heart disease. Current dietary strategies for the prevention of cardiovascular disease advocate adherence to low-fat/low-saturated-fat diets. Although there is no doubt that, in experimental conditions, low-fat diets offer an effective means of reducing blood cholesterol concentrations on a population basis, these appear to be less effective, largely due to poor compliance, attributed to low palatability and acceptability of these diets to the consumers. Due to the low consumer compliance, attempts have been made to identify other dietary components that can reduce blood cholesterol levels. Supplementation of diet with fermented dairy products or lactic acid bacteria containing dairy products has shown the potential to reduce serum cholesterol levels. Various approaches have been used to alleviate this issue, including the use of probiotics, especially Bifidobacterium spp. and Lactobacillus spp.. Probiotics, the living microorganisms that confer health benefits on the host when administered in adequate amounts, have received much attention on their proclaimed health benefits which include improvement in lactose intolerance, increase in natural resistance to infectious disease in gastrointestinal tract, suppression of cancer, antidiabetic, reduction in serum cholesterol level, and improved digestion. In addition, there are numerous reports on cholesterol removal ability of probiotics and their hypocholesterolemic effects. Several possible mechanisms for cholesterol removal by probiotics are assimilation of cholesterol by growing cells, binding of cholesterol to cellular surface, incorporation of

  20. Cholesterol-Lowering Probiotics as Potential Biotherapeutics for Metabolic Diseases

    Directory of Open Access Journals (Sweden)

    Manoj Kumar

    2012-01-01

    Full Text Available Cardiovascular diseases are one of the major causes of deaths in adults in the western world. Elevated levels of certain blood lipids have been reported to be the principal cause of cardiovascular disease and other disabilities in developed countries. Several animal and clinical trials have shown a positive association between cholesterol levels and the risks of coronary heart disease. Current dietary strategies for the prevention of cardiovascular disease advocate adherence to low-fat/low-saturated-fat diets. Although there is no doubt that, in experimental conditions, low-fat diets offer an effective means of reducing blood cholesterol concentrations on a population basis, these appear to be less effective, largely due to poor compliance, attributed to low palatability and acceptability of these diets to the consumers. Due to the low consumer compliance, attempts have been made to identify other dietary components that can reduce blood cholesterol levels. Supplementation of diet with fermented dairy products or lactic acid bacteria containing dairy products has shown the potential to reduce serum cholesterol levels. Various approaches have been used to alleviate this issue, including the use of probiotics, especially Bifidobacterium spp. and Lactobacillus spp.. Probiotics, the living microorganisms that confer health benefits on the host when administered in adequate amounts, have received much attention on their proclaimed health benefits which include improvement in lactose intolerance, increase in natural resistance to infectious disease in gastrointestinal tract, suppression of cancer, antidiabetic, reduction in serum cholesterol level, and improved digestion. In addition, there are numerous reports on cholesterol removal ability of probiotics and their hypocholesterolemic effects. Several possible mechanisms for cholesterol removal by probiotics are assimilation of cholesterol by growing cells, binding of cholesterol to cellular surface

  1. Analysis of gene expression levels in individual bacterial cells without image segmentation

    Energy Technology Data Exchange (ETDEWEB)

    Kwak, In Hae; Son, Minjun [Physics Department, University of Florida, P.O. Box 118440, Gainesville, FL 32611-8440 (United States); Hagen, Stephen J., E-mail: sjhagen@ufl.edu [Physics Department, University of Florida, P.O. Box 118440, Gainesville, FL 32611-8440 (United States)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer We present a method for extracting gene expression data from images of bacterial cells. Black-Right-Pointing-Pointer The method does not employ cell segmentation and does not require high magnification. Black-Right-Pointing-Pointer Fluorescence and phase contrast images of the cells are correlated through the physics of phase contrast. Black-Right-Pointing-Pointer We demonstrate the method by characterizing noisy expression of comX in Streptococcus mutans. -- Abstract: Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on a segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly.

  2. Analysis of gene expression levels in individual bacterial cells without image segmentation

    International Nuclear Information System (INIS)

    Kwak, In Hae; Son, Minjun; Hagen, Stephen J.

    2012-01-01

    Highlights: ► We present a method for extracting gene expression data from images of bacterial cells. ► The method does not employ cell segmentation and does not require high magnification. ► Fluorescence and phase contrast images of the cells are correlated through the physics of phase contrast. ► We demonstrate the method by characterizing noisy expression of comX in Streptococcus mutans. -- Abstract: Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on a segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly.

  3. Stable Regulation of Cell Cycle Events in Mycobacteria: Insights From Inherently Heterogeneous Bacterial Populations

    Science.gov (United States)

    Logsdon, Michelle M.; Aldridge, Bree B.

    2018-01-01

    Model bacteria, such as E. coli and B. subtilis, tightly regulate cell cycle progression to achieve consistent cell size distributions and replication dynamics. Many of the hallmark features of these model bacteria, including lateral cell wall elongation and symmetric growth and division, do not occur in mycobacteria. Instead, mycobacterial growth is characterized by asymmetric polar growth and division. This innate asymmetry creates unequal birth sizes and growth rates for daughter cells with each division, generating a phenotypically heterogeneous population. Although the asymmetric growth patterns of mycobacteria lead to a larger variation in birth size than typically seen in model bacterial populations, the cell size distribution is stable over time. Here, we review the cellular mechanisms of growth, division, and cell cycle progression in mycobacteria in the face of asymmetry and inherent heterogeneity. These processes coalesce to control cell size. Although Mycobacterium smegmatis and Mycobacterium bovis Bacillus Calmette-Guérin (BCG) utilize a novel model of cell size control, they are similar to previously studied bacteria in that initiation of DNA replication is a key checkpoint for cell division. We compare the regulation of DNA replication initiation and strategies used for cell size homeostasis in mycobacteria and model bacteria. Finally, we review the importance of cellular organization and chromosome segregation relating to the physiology of mycobacteria and consider how new frameworks could be applied across the wide spectrum of bacterial diversity. PMID:29619019

  4. Asynchrony in the growth and motility responses to environmental changes by individual bacterial cells

    International Nuclear Information System (INIS)

    Umehara, Senkei; Hattori, Akihiro; Inoue, Ippei; Yasuda, Kenji

    2007-01-01

    Knowing how individual cells respond to environmental changes helps one understand phenotypic diversity in a bacterial cell population, so we simultaneously monitored the growth and motility of isolated motile Escherichia coli cells over several generations by using a method called on-chip single-cell cultivation. Starved cells quickly stopped growing but remained motile for several hours before gradually becoming immotile. When nutrients were restored the cells soon resumed their growth and proliferation but remained immotile for up to six generations. A flagella visualization assay suggested that deflagellation underlies the observed loss of motility. This set of results demonstrates that single-cell transgenerational study under well-characterized environmental conditions can provide information that will help us understand distinct functions within individual cells

  5. Hijacking host cell highways: manipulation of the host actin cytoskeleton by obligate intracellular bacterial pathogens

    Directory of Open Access Journals (Sweden)

    Punsiri M Colonne

    2016-09-01

    Full Text Available Intracellular bacterial pathogens replicate within eukaryotic cells and display unique adaptations that support key infection events including invasion, replication, immune evasion, and dissemination. From invasion to dissemination, all stages of the intracellular bacterial life cycle share the same three-dimensional cytosolic space containing the host cytoskeleton. For successful infection and replication, many pathogens hijack the cytoskeleton using effector proteins introduced into the host cytosol by specialized secretion systems. A subset of effectors contains eukaryotic-like motifs that mimic host proteins to exploit signaling and modify specific cytoskeletal components such as actin and microtubules. Cytoskeletal rearrangement promotes numerous events that are beneficial to the pathogen, including internalization of bacteria, subversion of cell intrinsic immunity, structural support for bacteria-containing vacuoles, altered vesicular trafficking, actin-dependent bacterial movement, and pathogen dissemination. This review highlights a diverse group of obligate intracellular bacterial pathogens that manipulate the host cytoskeleton to thrive within eukaryotic cells and discusses underlying molecular mechanisms that promote these dynamic host-pathogen interactions.

  6. Purification of transfection-grade plasmid DNA from bacterial cells with superparamagnetic nanoparticles

    Science.gov (United States)

    Chiang, Chen-Li; Sung, Ching-Shan

    2006-07-01

    The functionalized magnetic nanobeads were used to develop a rapid protocol for extracting and purifying transfection-grade plasmid DNA from bacterial culture. Nanosized superparamagnetic nanoparticles (Fe 3O 4) were prepared by chemical coprecipitation method using Fe 2+, Fe 3+ salt, and ammonium hydroxide under a nitrogen atmosphere. The surface of Fe 3O 4 nanoparticles was modified by coating with the multivalent cationic agent, polyethylenimine (PEI). The PEI-modified magnetic nanobeads were employed to simplify the purification of plasmid DNA from bacterial cells. We demonstrated a useful plasmid, pRSETB-EGFP, encoding the green fluorescent protein with T7 promoter, was amplified in DE3 strain of Escherichia coli. The loaded nanobeads are recovered by magnetically driven separation and regenerated by exposure to the elution buffer with optimal ionic strength (1.25 M) and pH (9.0). Up to approximately 819 μg of high-purity (A 260/A 280 ratio=1.86) plasmid DNA was isolated from 100 ml of overnight bacterial culture. The eluted plasmid DNA was used directly for restriction enzyme digestion, bacterial cell transformation and animal cell transfection applications with success. The PEI-modified magnetic nanobead delivers significant time-savings, overall higher yields and better transfection efficiencies compared to anion-exchange and other methods. The results presented in this report show that PEI-modified magnetic nanobeads are suitable for isolation and purification of transfection-grade plasmid DNA.

  7. Chemical communication of antibiotic resistance by a highly resistant subpopulation of bacterial cells.

    Directory of Open Access Journals (Sweden)

    Omar M El-Halfawy

    Full Text Available The overall antibiotic resistance of a bacterial population results from the combination of a wide range of susceptibilities displayed by subsets of bacterial cells. Bacterial heteroresistance to antibiotics has been documented for several opportunistic Gram-negative bacteria, but the mechanism of heteroresistance is unclear. We use Burkholderia cenocepacia as a model opportunistic bacterium to investigate the implications of heterogeneity in the response to the antimicrobial peptide polymyxin B (PmB and also other bactericidal antibiotics. Here, we report that B. cenocepacia is heteroresistant to PmB. Population analysis profiling also identified B. cenocepacia subpopulations arising from a seemingly homogenous culture that are resistant to higher levels of polymyxin B than the rest of the cells in the culture, and can protect the more sensitive cells from killing, as well as sensitive bacteria from other species, such as Pseudomonas aeruginosa and Escherichia coli. Communication of resistance depended on upregulation of putrescine synthesis and YceI, a widely conserved low-molecular weight secreted protein. Deletion of genes for the synthesis of putrescine and YceI abrogate protection, while pharmacologic inhibition of putrescine synthesis reduced resistance to polymyxin B. Polyamines and YceI were also required for heteroresistance of B. cenocepacia to various bactericidal antibiotics. We propose that putrescine and YceI resemble "danger" infochemicals whose increased production by a bacterial subpopulation, becoming more resistant to bactericidal antibiotics, communicates higher level of resistance to more sensitive members of the population of the same or different species.

  8. Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

    Science.gov (United States)

    Muir, Elizabeth; Raza, Mansoor; Ellis, Clare; Burnside, Emily; Love, Fiona; Heller, Simon; Elliot, Matthew; Daniell, Esther; Dasgupta, Debayan; Alves, Nuno; Day, Priscilla; Fawcett, James; Keynes, Roger

    2017-01-01

    There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location. To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate. Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for

  9. Bacterial Ghosts as antigen and drug delivery system for ocular surface diseases: Effective internalization of Bacterial Ghosts by human conjunctival epithelial cells.

    Science.gov (United States)

    Kudela, Pavol; Koller, Verena Juliana; Mayr, Ulrike Beate; Nepp, Johannes; Lubitz, Werner; Barisani-Asenbauer, Talin

    2011-05-20

    The purpose of the presented investigation was to examine the efficiency of the novel carrier system Bacterial Ghosts (BGs), which are empty bacterial cell envelopes of Gram-negative bacteria to target human conjunctival epithelial cells, as well as to test the endocytic capacity of conjunctival cells after co-incubation with BGs generated from different bacterial species, and to foreclose potential cytotoxic effects caused by BGs. The efficiency of conjunctival cells to internalize BGs was investigated using the Chang conjunctival epithelial cell line and primary human conjunctiva-derived epithelial cells (HCDECs) as in vitro model. A high capacity of HCDECs to functionally internalize BGs was detected with the level of internalization depending on the type of species used for BGs generation. Detailed analysis showed no cytotoxic effect of BGs on HCDECs independently of the used bacterial species. Moreover, co-incubation with BGs did not enhance expression of both MHC class I and class II molecules by HCDECs, but increased expression of ICAM-1. The high rates of BG's internalization by HCDECs with no BG-mediated cytotoxic impact designate this carrier system to be a promising candidate for an ocular surface drug delivery system. BGs could be useful for future therapeutic ocular surface applications and eye-specific disease vaccine development including DNA transfer. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. A central role for carbon-overflow pathways in the modulation of bacterial cell death.

    Directory of Open Access Journals (Sweden)

    Vinai Chittezham Thomas

    2014-06-01

    Full Text Available Similar to developmental programs in eukaryotes, the death of a subpopulation of cells is thought to benefit bacterial biofilm development. However mechanisms that mediate a tight control over cell death are not clearly understood at the population level. Here we reveal that CidR dependent pyruvate oxidase (CidC and α-acetolactate synthase/decarboxylase (AlsSD overflow metabolic pathways, which are active during staphylococcal biofilm development, modulate cell death to achieve optimal biofilm biomass. Whereas acetate derived from CidC activity potentiates cell death in cells by a mechanism dependent on intracellular acidification and respiratory inhibition, AlsSD activity effectively counters CidC action by diverting carbon flux towards neutral rather than acidic byproducts and consuming intracellular protons in the process. Furthermore, the physiological features that accompany metabolic activation of cell death bears remarkable similarities to hallmarks of eukaryotic programmed cell death, including the generation of reactive oxygen species and DNA damage. Finally, we demonstrate that the metabolic modulation of cell death not only affects biofilm development but also biofilm-dependent disease outcomes. Given the ubiquity of such carbon overflow pathways in diverse bacterial species, we propose that the metabolic control of cell death may be a fundamental feature of prokaryotic development.

  11. Cytolethal distending toxin (CDT): a bacterial weapon to control host cell proliferation?

    Science.gov (United States)

    De Rycke, J; Oswald, E

    2001-09-25

    Cytolethal distending toxins (CDT) constitute a family of genetically related bacterial protein toxins able to stop the proliferation of numerous cell lines. This effect is due to their ability to trigger in target cells a signaling pathway that normally prevents the transition between the G2 and the M phase of the cell cycle. Produced by several unrelated Gram-negative mucosa-associated bacterial species, CDTs are determined by a cluster of three adjacent genes (cdtA, cdtB, cdtC) encoding proteins whose respective role is not yet fully elucidated. The CDT-B protein presents sequence homology to several mammalian and bacterial phosphodiesterases, such as DNase I. The putative nuclease activity of CDT-B, together with the activation by CDT of a G2 cell cycle checkpoint, strongly suggests that CDT induces an as yet uncharacterized DNA alteration. However, the effective entry of CDT into cells and subsequent translocation into the nucleus have not yet been demonstrated by direct methods. The relationship between the potential DNA-damaging properties of this original family of toxins and their role as putative virulence factors is discussed.

  12. Cell-to-cell variation and specialization in sugar metabolism in clonal bacterial populations

    OpenAIRE

    Nikolic, Nela; Schreiber, Frank; Dal Co, Alma; Kiviet, Daniel J.; Bergmiller, Tobias; Littmann, Sten; Kuypers, Marcel M. M.; Ackermann, Martin

    2017-01-01

    Author summary This study addresses a fundamental question in bacterial metabolism: do all individuals in a clonal population express the same metabolic functions, or do individuals specialize on different metabolic functions and assimilate different substrates? Reports about stochastic gene expression in bacterial populations raise the possibility that transcriptional differences between individuals translate into different metabolic behaviors, but the prevalence and magnitude of such effect...

  13. Targeted ion parking for the quantitation of biotherapeutic proteins: concepts and preliminary data.

    Science.gov (United States)

    Campbell, J Larry; Le Blanc, J C Yves

    2010-12-01

    Targeted ion parking (or TIPing) is the first quantitative application of ion/ion reactions for mass spectrometry. In TIPing, intact biotherapeutic proteins are electrosprayed as intact molecules (no digestion) and, as expected, many multiply protonated species are produced (e.g., (M + 7H)(7+), (M + 8H)(8+), etc.). Several of these multiply charged species are selectively isolated using a quadrupole mass analyzer and then contained in a linear ion trap. The protein ions are then subjected to a proton-transfer reaction with a reagent anion. The ions undergo sequential charge reduction (e.g., to (M + 6H)(6+)) during a defined reaction period. Applying a low-amplitude waveform to the trap during this reaction time stops the ion/ion reaction at a chosen (and predicted) charge state for the protein. This funnels the analyte ions into a single channel with relatively high efficiency (>50% of reactant ion signal is converted into product ion signal) that can be used for quantitation. In TIPing, the target protein's molecular weight and charge state distribution are the only prerequisite knowledge required. This information can be acquired experimentally or can be easily predicted based upon amino acid sequences. Preliminary data for a biotherapeutic protein, a domain antibody, were collected using TIPing coupled online with liquid chromatography (LC-TIPing). The LC-TIPing data demonstrate a linear response for samples from 10-1000 ng/mL extracted from a complex plasma sample, demonstrating the analytical potential for TIPing. Copyright © 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.

  14. Effect of biotherapeutics on antitoxin IgG in experimentally induced Clostridium difficile infection

    Directory of Open Access Journals (Sweden)

    S Kaur

    2012-01-01

    Full Text Available Purpose: Recurrent diarrhoea after successful treatment of primary Clostridium difficile associated disease (CDAD occurs due to bowel flora alterations and failure to mount an effective antibody response. Apart from antibiotics, risk factors include immunosuppressive and acid-suppressive drug administration. Biotherapeutics such as probiotic and epidermal growth factor (EGF may offer potential effective therapy for CDAD. Materials and Methods: The effect of biotherapeutics in mounting an antibody response against C. difficile toxins was studied in BALB/c mice challenged with C. difficile after pre-treatment with ampicillin, lansoprazole or cyclosporin. Sera from sacrificed animals were estimated for antitoxin IgG by enzyme linked immunosorbent assay. Results: Antitoxin IgG was significantly higher (P0.05 in animals in which C. difficile was given after pre-treatment with cyclosporin compared to those without any pre-treatment, or pre-treatment with antibiotic or lansoprazole. In inter-subgroup comparisons also significant anomaly in production of antitoxin IgG was found. The antitoxin IgG levels were raised in animals administered C. difficile after pre-treatment with ampicillin, but lower in animals administered cyclosporin. High levels of antitoxin IgG were also found in the serum samples of animals receiving lansoprazole and C. difficile. Conclusions: Probiotics showed their beneficial effect by boosting the immune response as seen by production of antitoxin IgG. Oral administration of EGF did not affect the immune response to C. difficile toxins as significant increase was not observed in the serum antitoxin IgG levels in any of the groups investigated.

  15. Structural constraints and dynamics of bacterial cell wall architecture

    Directory of Open Access Journals (Sweden)

    Miguel Angel De Pedro

    2015-05-01

    Full Text Available The peptidoglycan wall (PG is a unique structure which confers physical strength and defined shape to bacteria. It consists of a net-like macromolecule of peptide interlinked glycan chains overlying the cell membrane. The structure and layout of the PG dictates that the wall has to be continuously modified as bacteria go through division, morphological differentiation and adaptive responses. The PG is poorly known in structural terms. However, to understand morphogenesis a precise knowledge of glycan strand arrangement and of local effects of the different kinds of subunits is essential. The scarcity of data led to a conception of the PG as a regular, highly ordered structure which strongly influenced growth models. Here, we review the structure of the PG to define a more realistic conceptual framework. We discuss the consequences of the plasticity of murein architecture in morphogenesis and try to define a set of minimal structural constraints that must be fulfilled by any model to be compatible with present day information.

  16. Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing

    OpenAIRE

    J. Riba; T. Gleichmann; S. Zimmermann; R. Zengerle; P. Koltay

    2016-01-01

    The isolation and analysis of single prokaryotic cells down to 1??m and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35?pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20??m in size....

  17. Plectasin, a Fungal Defensin, Targets the Bacterial Cell Wall Precursor Lipid II

    DEFF Research Database (Denmark)

    Schneider, Tanja; Kruse, Thomas; Wimmer, Reinhard

    2010-01-01

    Host defense peptides such as defensins are components of innate immunity and have retained antibiotic activity throughout evolution. Their activity is thought to be due to amphipathic structures, which enable binding and disruption of microbial cytoplasmic membranes. Contrary to this, we show...... that plectasin, a fungal defensin, acts by directly binding the bacterial cell-wall precursor Lipid II. A wide range of genetic and biochemical approaches identify cell-wall biosynthesis as the pathway targeted by plectasin. In vitro assays for cell-wall synthesis identified Lipid II as the specific cellular...

  18. Live Staphylococcus aureus and bacterial soluble factors induce different transcriptional responses in human airway cells.

    Science.gov (United States)

    Moreilhon, Chimène; Gras, Delphine; Hologne, Coralie; Bajolet, Odile; Cottrez, Françoise; Magnone, Virginie; Merten, Marc; Groux, Hervé; Puchelle, Edith; Barbry, Pascal

    2005-02-10

    To characterize the response of respiratory epithelium to infection by Staphylococcus aureus (S. aureus), human airway cells were incubated for 1 to 24 h with a supernatant of a S. aureus culture (bacterial supernatant), then profiled with a pangenomic DNA microarray. Because an upregulation of many genes was noticed around 3 h, three independent approaches were then used to characterize the host response to a 3-h contact either with bacterial supernatant or with live bacteria: 1) a DNA microarray containing 4,200 sequence-verified probes, 2) a semiquantitative RT-PCR with a set of 537 pairs of validated primers, or 3) ELISA assay of IL-8, IL-6, TNFalpha, and PGE(2). Among others, Fos, Jun, and EGR-1 were upregulated by the bacterial supernatant and by live bacteria. Increased expression of bhlhb2 and Mig-6, promoter regions which harbor HIF responding elements, was explained by an increased expression of the HIF-1alpha protein. Activation of the inducible form of cyclooxygenase, COX-2, and of the interleukins IL-1, IL-6, and IL-8, as well as of the NF-kappaB pathway, was observed preferentially in cells in contact with bacterial supernatant. Early infection was characterized by an upregulation of anti-apoptotic genes and a downregulation of pro-apoptotic genes. This correlated with a necrotic, rather than apoptotic cell death. Overall, this first global description of an airway epithelial infection by S. aureus demonstrates a larger global response to bacterial supernatant (in term of altered genes and variation factors) than to exponentially growing live bacteria.

  19. Flow cytometric bacterial cell counts challenge conventional heterotrophic plate counts for routine microbiological drinking water monitoring

    KAUST Repository

    Van Nevel, S.

    2017-02-08

    Drinking water utilities and researchers continue to rely on the century-old heterotrophic plate counts (HPC) method for routine assessment of general microbiological water quality. Bacterial cell counting with flow cytometry (FCM) is one of a number of alternative methods that challenge this status quo and provide an opportunity for improved water quality monitoring. After more than a decade of application in drinking water research, FCM methodology is optimised and established for routine application, supported by a considerable amount of data from multiple full-scale studies. Bacterial cell concentrations obtained by FCM enable quantification of the entire bacterial community instead of the minute fraction of cultivable bacteria detected with HPC (typically < 1% of all bacteria). FCM measurements are reproducible with relative standard deviations below 3% and can be available within 15 min of samples arriving in the laboratory. High throughput sample processing and complete automation are feasible and FCM analysis is arguably less expensive than HPC when measuring more than 15 water samples per day, depending on the laboratory and selected staining procedure(s). Moreover, many studies have shown FCM total (TCC) and intact (ICC) cell concentrations to be reliable and robust process variables, responsive to changes in the bacterial abundance and relevant for characterising and monitoring drinking water treatment and distribution systems. The purpose of this critical review is to initiate a constructive discussion on whether FCM could replace HPC in routine water quality monitoring. We argue that FCM provides a faster, more descriptive and more representative quantification of bacterial abundance in drinking water.

  20. Convergent development of anodic bacterial communities in microbial fuel cells.

    KAUST Repository

    Yates, Matthew D

    2012-05-10

    Microbial fuel cells (MFCs) are often inoculated from a single wastewater source. The extent that the inoculum affects community development or power production is unknown. The stable anodic microbial communities in MFCs were examined using three inocula: a wastewater treatment plant sample known to produce consistent power densities, a second wastewater treatment plant sample, and an anaerobic bog sediment. The bog-inoculated MFCs initially produced higher power densities than the wastewater-inoculated MFCs, but after 20 cycles all MFCs on average converged to similar voltages (470±20 mV) and maximum power densities (590±170 mW m(-2)). The power output from replicate bog-inoculated MFCs was not significantly different, but one wastewater-inoculated MFC (UAJA3 (UAJA, University Area Joint Authority Wastewater Treatment Plant)) produced substantially less power. Denaturing gradient gel electrophoresis profiling showed a stable exoelectrogenic biofilm community in all samples after 11 cycles. After 16 cycles the predominance of Geobacter spp. in anode communities was identified using 16S rRNA gene clone libraries (58±10%), fluorescent in-situ hybridization (FISH) (63±6%) and pyrosequencing (81±4%). While the clone library analysis for the underperforming UAJA3 had a significantly lower percentage of Geobacter spp. sequences (36%), suggesting that a predominance of this microbe was needed for convergent power densities, the lower percentage of this species was not verified by FISH or pyrosequencing analyses. These results show that the predominance of Geobacter spp. in acetate-fed systems was consistent with good MFC performance and independent of the inoculum source.

  1. The interaction of bacterial magnetosomes and human liver cancer cells in vitro

    Science.gov (United States)

    Wang, Pingping; Chen, Chuanfang; Chen, Changyou; Li, Yue; Pan, Weidong; Song, Tao

    2017-04-01

    As the biogenic magnetic nanomaterial, bacterial magnetic nanoparticles, namely magnetosomes, provide many advantages for potential biomedical applications. As such, interactions among magnetosomes and target cells should be elucidated to develop their bioapplications and evaluate their biocompatibilities. In this study, the interaction of magnetosomes and human liver cancer HepG2 cells was examined. Prussian blue staining revealed numerous stained particles in or on the cells. Intracellular iron concentrations, measured through inductively coupled plasma optical emission spectroscopy, increased with the increasing concentration of the magnetosomes. Transmission electron microscopy images showed that magnetosomes could be internalized in cells, mainly encapsulated in membrane vesicles, such as endosomes and lysosomes, and partly found as free particles in the cytosol. Some of the magnetosomes on cellular surfaces were encapsulated through cell membrane ruffling, which is the initiating process of endocytosis. Applying low temperature treatment and using specific endocytic inhibitors, we validated that macropinocytosis and clathrin-mediated endocytosis were involved in magnetosome uptake by HepG2 cells. Consequently, we revealed the interaction and intrinsic endocytic mechanisms of magnetosomes and HepG2 cells. This study provides a basis for the further research on bacterial magnetosome applications in liver diseases.

  2. Biomechanical loading modulates proinflammatory and bone resorptive mediators in bacterial-stimulated PDL cells.

    Science.gov (United States)

    Nogueira, Andressa Vilas Boas; Nokhbehsaim, Marjan; Eick, Sigrun; Bourauel, Christoph; Jäger, Andreas; Jepsen, Søren; Rossa, Carlos; Deschner, James; Cirelli, Joni Augusto

    2014-01-01

    The present study aimed to evaluate in vitro whether biomechanical loading modulates proinflammatory and bone remodeling mediators production by periodontal ligament (PDL) cells in the presence of bacterial challenge. Cells were seeded on BioFlex culture plates and exposed to Fusobacterium nucleatum ATCC 25586 and/or cyclic tensile strain (CTS) of low (CTSL) and high (CTSH) magnitudes for 1 and 3 days. Synthesis of cyclooxygenase-2 (COX2) and prostaglandin E2 (PGE2) was evaluated by ELISA. Gene expression and protein secretion of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were evaluated by quantitative RT-PCR and ELISA, respectively. F. nucleatum increased the production of COX2 and PGE2, which was further increased by CTS. F. nucleatum-induced increase of PGE2 synthesis was significantly (P orthodontic or occlusal, loading may enhance the bacterial-induced inflammation and destruction in periodontitis.

  3. Regulation of bacterial virulence gene expression by cell envelope stress responses.

    Science.gov (United States)

    Flores-Kim, Josué; Darwin, Andrew J

    2014-01-01

    The bacterial cytoplasm lies within a multilayered envelope that must be protected from internal and external hazards. This protection is provided by cell envelope stress responses (ESRs), which detect threats and reprogram gene expression to ensure survival. Pathogens frequently need these ESRs to survive inside the host, where their envelopes face dangerous environmental changes and attack from antimicrobial molecules. In addition, some virulence genes have become integrated into ESR regulons. This might be because these genes can protect the cell envelope from damage by host molecules, or it might help ESRs to reduce stress by moderating the assembly of virulence factors within the envelope. Alternatively, it could simply be a mechanism to coordinate the induction of virulence gene expression with entry into the host. Here, we briefly describe some of the bacterial ESRs, followed by examples where they control virulence gene expression in both Gram-negative and Gram-positive pathogens.

  4. The influence of radiation on bacterial cells and their proteolytic properties

    International Nuclear Information System (INIS)

    Szulc, M.; Stefaniakowa, A.; Stanczak, B.; Peconek, J.

    1980-01-01

    The suspension of bacterial cells and their spores were exposed to X rays in the environment with and without protein. The doses of radiation ranged from 1 to 100 Gy and in case of spores of B. subtilis from 50 to 1000 Gy. It was found that irradiation to Proteus vulgaris, Pseudomonas fluorescens and Ps. aeruginosa caused an inconsiderable decrease of proteolytic properties of the generation originated from irradiated bacteria. Irradiation of B. subtilis spores did not influence the proteolytic activity of bacterial cells derived from the exposed spores. The degree of wasting away of bacteria exposed to the same radiation was higher than the rate of proteolytic properties decrease. The presence of protein in the surroundings had no influence on proteolytic characteristics of new generations. (author)

  5. Influenza viral neuraminidase primes bacterial coinfection through TGF-β-mediated expression of host cell receptors.

    Science.gov (United States)

    Li, Ning; Ren, Aihui; Wang, Xiaoshuang; Fan, Xin; Zhao, Yong; Gao, George F; Cleary, Patrick; Wang, Beinan

    2015-01-06

    Influenza infection predisposes the host to secondary bacterial pneumonia, which is a major cause of mortality during influenza epidemics. The molecular mechanisms underlying the bacterial coinfection remain elusive. Neuraminidase (NA) of influenza A virus (IAV) enhances bacterial adherence and also activates TGF-β. Because TGF-β can up-regulate host adhesion molecules such as fibronectin and integrins for bacterial binding, we hypothesized that activated TGF-β during IAV infection contributes to secondary bacterial infection by up-regulating these host adhesion molecules. Flow cytometric analyses of a human lung epithelial cell line indicated that the expression of fibronectin and α5 integrin was up-regulated after IAV infection or treatment with recombinant NA and was reversed through the inhibition of TGF-β signaling. IAV-promoted adherence of group A Streptococcus (GAS) and other coinfective pathogens that require fibronectin for binding was prevented significantly by the inhibition of TGF-β. However, IAV did not promote the adherence of Lactococcus lactis unless this bacterium expressed the fibronectin-binding protein of GAS. Mouse experiments showed that IAV infection enhanced GAS colonization in the lungs of wild-type animals but not in the lungs of mice deficient in TGF-β signaling. Taken together, these results reveal a previously unrecognized mechanism: IAV NA enhances the expression of cellular adhesins through the activation of TGF-β, leading to increased bacterial loading in the lungs. Our results suggest that TGF-β and cellular adhesins may be potential pharmaceutical targets for the prevention of coinfection.

  6. Quantifying bacterial adhesion on antifouling polymer brushes via single-cell force spectroscopy

    Czech Academy of Sciences Publication Activity Database

    Rodriguez-Emmenegger, Cesar; Janel, S.; de los Santos Pereira, Andres; Bruns, M.; Lafont, F.

    2015-01-01

    Roč. 6, č. 31 (2015), s. 5740-5751 ISSN 1759-9954 R&D Projects: GA ČR(CZ) GJ15-09368Y; GA MŠk(CZ) ED1.1.00/02.0109 Grant - others:OPPK(XE) CZ.2.16/3.1.00/21545 Program:OPPK Institutional support: RVO:61389013 Keywords : antifouling polymer brushes * single-cell force spectroscopy * bacterial adhesion Subject RIV: BO - Biophysics Impact factor: 5.687, year: 2015

  7. Instrumental analysis of bacterial cells using vibrational and emission Moessbauer spectroscopic techniques

    Energy Technology Data Exchange (ETDEWEB)

    Kamnev, Alexander A. [Laboratory of Biochemistry of Plant-Bacterial Symbioses, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 410049 Saratov (Russian Federation)]. E-mail: aakamnev@ibppm.sgu.ru; Tugarova, Anna V. [Laboratory of Biochemistry of Plant-Bacterial Symbioses, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 410049 Saratov (Russian Federation); Antonyuk, Lyudmila P. [Laboratory of Biochemistry of Plant-Bacterial Symbioses, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 410049 Saratov (Russian Federation); Tarantilis, Petros A. [Laboratory of Chemistry, Department of Science, Agricultural University of Athens, 11855 Athens (Greece); Kulikov, Leonid A. [Laboratory of Nuclear Chemistry Techniques, Department of Radiochemistry, Faculty of Chemistry, M.V. Lomonosov Moscow State University, 119992 Moscow (Russian Federation); Perfiliev, Yurii D. [Laboratory of Nuclear Chemistry Techniques, Department of Radiochemistry, Faculty of Chemistry, M.V. Lomonosov Moscow State University, 119992 Moscow (Russian Federation); Polissiou, Moschos G. [Laboratory of Chemistry, Department of Science, Agricultural University of Athens, 11855 Athens (Greece); Gardiner, Philip H.E. [Division of Chemistry, School of Science and Mathematics, Sheffield Hallam University, Sheffield S1 1WB (United Kingdom)

    2006-07-28

    In biosciences and biotechnology, the expanding application of physicochemical approaches using modern instrumental techniques is an efficient strategy to obtain valuable and often unique information at the molecular level. In this work, we applied a combination of vibrational (Fourier transform infrared (FTIR), FT-Raman) spectroscopic techniques, useful in overall structural and compositional analysis of bacterial cells of the rhizobacterium Azospirillum brasilense, with {sup 57}Co emission Moessbauer spectroscopy (EMS) used for sensitive monitoring of metal binding and further transformations in live bacterial cells. The information obtained, together with ICP-MS analyses for metals taken up by the bacteria, is useful in analysing the impact of the environmental conditions (heavy metal stress) on the bacterial metabolism and some differences in the heavy metal stress-induced behaviour of non-endophytic (Sp7) and facultatively endophytic (Sp245) strains. The results show that, while both strains Sp7 and Sp245 take up noticeable and comparable amounts of heavy metals from the medium (0.12 and 0.13 mg Co, 0.48 and 0.44 mg Cu or 4.2 and 2.1 mg Zn per gram of dry biomass, respectively, at a metal concentration of 0.2 mM in the medium), their metabolic responses differ essentially. Whereas for strain Sp7 the FTIR measurements showed significant accumulation of polyhydroxyalkanoates as storage materials involved in stress endurance, strain Sp245 did not show any major changes in cellular composition. Nevertheless, EMS measurements showed rapid binding of cobalt(II) by live bacterial cells (chemically similar to metal binding by dead bacteria) and its further transformation in the live cells within an hour.

  8. SLiCE: a novel bacterial cell extract-based DNA cloning method

    OpenAIRE

    Zhang, Yongwei; Werling, Uwe; Edelmann, Winfried

    2012-01-01

    We describe a novel cloning method termed SLiCE (Seamless Ligation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional s...

  9. Arthrobacter Species as a Prey Cell Reservoir for Nonobligate Bacterial Predators in Soil

    Science.gov (United States)

    1989-01-01

    TUNC,.ASSIFIED MA4 i COPY - FOR REPRODUCTION PURPOSES IF:TrY CLASSIFICATION OF THIS PAGE REPORT DOCUMENTATION PAGE la . REPORT SECURITY CLASSIFICATION...unlimited. BER(S) S MONITORING ORGANIZATION REPORT NUMBER(S) ARO 22469.13-LS 6a. NAME OF PERFORMING ORGANIZATION 6b. OFFICE SYMBOL 7a. NAME OF MONITORING...species as a prey cell reservoir for nonobligate bacterial predators in soil. Can. J. Microbiol. 35 : 559--564, tine investigation at etc entreprise sur

  10. Tuning of glyconanomaterial shape and size for selective bacterial cell agglutination

    OpenAIRE

    Cid Martín, J.J.; Assali, Mohyeddin; Fernández García, E.; Valdivia Giménez, Victoria Esther; Sánchez Fernández, E. M.; García Fernández, José Manuel; Wellinger, Ralf Erik; Fernández Fernández, Inmaculada; Khiar, N.

    2016-01-01

    Multivalent glycosystems are potential candidates for anti-adhesive therapy, a non-lethal approach against the ever increasing antibiotic resistance of pathogenic bacteria. In order to fine-tune the glyconanomaterial size and shape for selective bacterial cell agglutination, herein we report the synthesis of sugar-coated dynamic and polymeric 3D-micelles and 1D-carbon nanotubes. The reported shot-gun like synthetic approach is based on the ability of diacetylenic-based neoglycolipids to self-...

  11. Radiosensitization of hypoxic bacterial cells and animal tumours by membrane active drugs and hyperthermia

    International Nuclear Information System (INIS)

    Singh, B.B.; Srinivasan, V.T.; Shenoy, M.A.; George, K.C.; Maniar, H.S.; Rawat, K.P.

    1987-01-01

    The present report deals with the results on phenothiazine derivatives such as promethazine (PMZ), trimeprazine (TMZ), trifluoperazine (TFP) and prochlorperazine (PCP) and their comparison with that of chlorpromazine (CPZ). Their efficiency in combination with hyperthermia, radiation and other anti-cancer drugs in treating murine tumors has also been presented herein. In addition, results on bacterial cells dealing with their mechanistic aspects are also included. (author). 57 refs., 27 figures, 13 tables

  12. Instrumental analysis of bacterial cells using vibrational and emission Moessbauer spectroscopic techniques

    International Nuclear Information System (INIS)

    Kamnev, Alexander A.; Tugarova, Anna V.; Antonyuk, Lyudmila P.; Tarantilis, Petros A.; Kulikov, Leonid A.; Perfiliev, Yurii D.; Polissiou, Moschos G.; Gardiner, Philip H.E.

    2006-01-01

    In biosciences and biotechnology, the expanding application of physicochemical approaches using modern instrumental techniques is an efficient strategy to obtain valuable and often unique information at the molecular level. In this work, we applied a combination of vibrational (Fourier transform infrared (FTIR), FT-Raman) spectroscopic techniques, useful in overall structural and compositional analysis of bacterial cells of the rhizobacterium Azospirillum brasilense, with 57 Co emission Moessbauer spectroscopy (EMS) used for sensitive monitoring of metal binding and further transformations in live bacterial cells. The information obtained, together with ICP-MS analyses for metals taken up by the bacteria, is useful in analysing the impact of the environmental conditions (heavy metal stress) on the bacterial metabolism and some differences in the heavy metal stress-induced behaviour of non-endophytic (Sp7) and facultatively endophytic (Sp245) strains. The results show that, while both strains Sp7 and Sp245 take up noticeable and comparable amounts of heavy metals from the medium (0.12 and 0.13 mg Co, 0.48 and 0.44 mg Cu or 4.2 and 2.1 mg Zn per gram of dry biomass, respectively, at a metal concentration of 0.2 mM in the medium), their metabolic responses differ essentially. Whereas for strain Sp7 the FTIR measurements showed significant accumulation of polyhydroxyalkanoates as storage materials involved in stress endurance, strain Sp245 did not show any major changes in cellular composition. Nevertheless, EMS measurements showed rapid binding of cobalt(II) by live bacterial cells (chemically similar to metal binding by dead bacteria) and its further transformation in the live cells within an hour

  13. Towards identifying host cell-type specific response patterns to bacterial endosymbiosis

    DEFF Research Database (Denmark)

    Gavrilovic, Srdjan

    the evolutionary success of the interaction. Precise spatial and temporal coordination of the expression of numerous genes is necessary to develop and maintain such a complex interaction. Many gene expression studies have been conducted to gain insight into bacterial root endosymbiosis. From a historical point......, and whole plant transformants were regenerated. These will form a basis for isolating transcriptionally active mRNA fractions associated with ribosomes and 21 nt long small RNAs from targeted cell populations....

  14. Magnetically modified bacterial cellulose: A promising carrier for immobilization of affinity ligands, enzymes, and cells

    Czech Academy of Sciences Publication Activity Database

    Baldíková, E.; Pospíšková, K.; Ladakis, D.; Kookos, I.K.; Koutinas, A.A.; Šafaříková, Miroslava; Šafařík, Ivo

    2017-01-01

    Roč. 71, February (2017), s. 214-221 ISSN 0928-4931 Institutional support: RVO:60077344 Keywords : bacterial cellulose * Komagataeibacter sucrofermentans * copper phthalocyanine * crystal violet * yeast cells * trypsin Subject RIV: EI - Biotechnology ; Bionics OBOR OECD: Bioproducts (products that are manufactured using biological material as feedstock) biomaterials, bioplastics, biofuels, bioderived bulk and fine chemicals, bio-derived novel materials Impact factor: 4.164, year: 2016

  15. Repeatability of differential goat bulk milk culture and associations with somatic cell count, total bacterial count, and standard plate count

    NARCIS (Netherlands)

    Koop, G.; Dik, N.; Nielen, M.; Lipman, L.J.A.

    2010-01-01

    The aims of this study were to assess how different bacterial groups in bulk milk are related to bulk milk somatic cell count (SCC), bulk milk total bacterial count (TBC), and bulk milk standard plate count (SPC) and to measure the repeatability of bulk milk culturing. On 53 Dutch dairy goat farms,

  16. Biomechanical Loading Modulates Proinflammatory and Bone Resorptive Mediators in Bacterial-Stimulated PDL Cells

    Directory of Open Access Journals (Sweden)

    Andressa Vilas Boas Nogueira

    2014-01-01

    Full Text Available The present study aimed to evaluate in vitro whether biomechanical loading modulates proinflammatory and bone remodeling mediators production by periodontal ligament (PDL cells in the presence of bacterial challenge. Cells were seeded on BioFlex culture plates and exposed to Fusobacterium nucleatum ATCC 25586 and/or cyclic tensile strain (CTS of low (CTSL and high (CTSH magnitudes for 1 and 3 days. Synthesis of cyclooxygenase-2 (COX2 and prostaglandin E2 (PGE2 was evaluated by ELISA. Gene expression and protein secretion of osteoprotegerin (OPG and receptor activator of nuclear factor kappa-B ligand (RANKL were evaluated by quantitative RT-PCR and ELISA, respectively. F. nucleatum increased the production of COX2 and PGE2, which was further increased by CTS. F. nucleatum-induced increase of PGE2 synthesis was significantly (P<0.05 increased when CTSH was applied at 1 and 3 days. In addition, CTSH inhibited the F. nucleatum-induced upregulation of OPG at 1 and 3 days, thereby increasing the RANKL/OPG ratio. OPG and RANKL mRNA results correlated with the protein results. In summary, our findings provide original evidence that CTS can enhance bacterial-induced syntheses of molecules associated with inflammation and bone resorption by PDL cells. Therefore, biomechanical, such as orthodontic or occlusal, loading may enhance the bacterial-induced inflammation and destruction in periodontitis.

  17. Spectrum of bacterial colonization associated with urothelial cells from patients with chronic lower urinary tract symptoms.

    Science.gov (United States)

    Khasriya, Rajvinder; Sathiananthamoorthy, Sanchutha; Ismail, Salim; Kelsey, Michael; Wilson, Mike; Rohn, Jennifer L; Malone-Lee, James

    2013-07-01

    Chronic lower urinary tract symptoms (LUTS), such as urgency and incontinence, are common, especially among the elderly, but their etiology is often obscure. Recent studies of acute urinary tract infections implicated invasion by Escherichia coli into the cytoplasm of urothelial cells, with persistence of long-term bacterial reservoirs, but the role of infection in chronic LUTS is unknown. We conducted a large prospective study with eligible patients with LUTS and controls over a 3-year period, comparing routine urine cultures of planktonic bacteria with cultures of shed urothelial cells concentrated in centrifuged urinary sediments. This comparison revealed large numbers of bacteria undetected by routine cultures. Next, we typed the bacterial species cultured from patient and control sediments under both aerobic and anaerobic conditions, and we found that the two groups had complex but significantly distinct profiles of bacteria associated with their shed bladder epithelial cells. Strikingly, E. coli, the organism most responsible for acute urinary tract infections, was not the only or even the main offending pathogen in this more-chronic condition. Antibiotic protection assays with shed patient cells and in vitro infection studies using patient-derived strains in cell culture suggested that LUTS-associated bacteria are within or extremely closely associated with shed epithelial cells, which explains how routine cultures might fail to detect them. These data have strong implications for the need to rethink our common diagnoses and treatments of chronic urinary tract symptoms.

  18. CMEIAS-Aided Microscopy of the Spatial Ecology of Individual Bacterial Interactions Involving Cell-to-Cell Communication within Biofilms

    Directory of Open Access Journals (Sweden)

    Frank B. Dazzo

    2012-05-01

    Full Text Available This paper describes how the quantitative analytical tools of CMEIAS image analysis software can be used to investigate in situ microbial interactions involving cell-to-cell communication within biofilms. Various spatial pattern analyses applied to the data extracted from the 2-dimensional coordinate positioning of individual bacterial cells at single-cell resolution indicate that microbial colonization within natural biofilms is not a spatially random process, but rather involves strong positive interactions between communicating cells that influence their neighbors’ aggregated colonization behavior. Geostatistical analysis of the data provide statistically defendable estimates of the micrometer scale and interpolation maps of the spatial heterogeneity and local intensity at which these microbial interactions autocorrelate with their spatial patterns of distribution. Including in situ image analysis in cell communication studies fills an important gap in understanding the spatially dependent microbial ecophysiology that governs the intensity of biofilm colonization and its unique architecture.

  19. CMEIAS-aided microscopy of the spatial ecology of individual bacterial interactions involving cell-to-cell communication within biofilms.

    Science.gov (United States)

    Dazzo, Frank B

    2012-01-01

    This paper describes how the quantitative analytical tools of CMEIAS image analysis software can be used to investigate in situ microbial interactions involving cell-to-cell communication within biofilms. Various spatial pattern analyses applied to the data extracted from the 2-dimensional coordinate positioning of individual bacterial cells at single-cell resolution indicate that microbial colonization within natural biofilms is not a spatially random process, but rather involves strong positive interactions between communicating cells that influence their neighbors' aggregated colonization behavior. Geostatistical analysis of the data provide statistically defendable estimates of the micrometer scale and interpolation maps of the spatial heterogeneity and local intensity at which these microbial interactions autocorrelate with their spatial patterns of distribution. Including in situ image analysis in cell communication studies fills an important gap in understanding the spatially dependent microbial ecophysiology that governs the intensity of biofilm colonization and its unique architecture.

  20. Temporal expression of bacterial proteins instructs host CD4 T cell expansion and Th17 development.

    Directory of Open Access Journals (Sweden)

    Seung-Joo Lee

    2012-01-01

    Full Text Available Pathogens can substantially alter gene expression within an infected host depending on metabolic or virulence requirements in different tissues, however, the effect of these alterations on host immunity are unclear. Here we visualized multiple CD4 T cell responses to temporally expressed proteins in Salmonella-infected mice. Flagellin-specific CD4 T cells expanded and contracted early, differentiated into Th1 and Th17 lineages, and were enriched in mucosal tissues after oral infection. In contrast, CD4 T cells responding to Salmonella Type-III Secretion System (TTSS effectors steadily accumulated until bacterial clearance was achieved, primarily differentiated into Th1 cells, and were predominantly detected in systemic tissues. Thus, pathogen regulation of antigen expression plays a major role in orchestrating the expansion, differentiation, and location of antigen-specific CD4 T cells in vivo.

  1. Application of silica magnetite nanocomposites to the isolation of ultrapure plasmid DNA from bacterial cells

    Science.gov (United States)

    Chiang, Chen-Li; Sung, Ching-Shan; Chen, Chuh-Yean

    2006-10-01

    The aim of this study was to develop a simple and rapid method for purification of ultrapure plasmid DNA with high yields from bacterial cultures. Nanosized superparamagnetic nanoparticles (Fe 3O 4) were prepared by chemical precipitation method using Fe 2+, Fe 3+ salt, and ammonium hydroxide under a nitrogen atmosphere. Silica-magnetite nanocomposites were prepared by the method of acid hydrolysis of tetraethoxysilane (TEOS) to coat the silica onto magnetite nanoparticles. DNA was adsorbed to the support under high salt conditions, and recovered directly in water for immediate downstream application, without the need for precipitation. We demonstrated that a useful plasmid, pRSETB-EGFP, encoding for the green fluorescent protein with T7 promoter, could be amplified in Escherichia coli of DE3 strain. Up to approximately 43 μg of high-purity ( A260/ A280 ratio=1.75) plasmid DNA was isolated from 3 ml of an overnight bacterial culture. The eluted plasmid DNA was used directly for restriction enzyme digestion, bacterial cell transformation and polymerase chain reaction (PCR) amplification with success. The protocol, starting from the preparation of bacterial lysate and ending with purified plasmid takes less than 8 min. The silica-magnetite nanocomposites deliver significant time-savings, overall higher yields, lower RNA contamination, and better PCR amplification compared to commercial available silica-based and other methods.

  2. Bacterial meningitis in hematopoietic stem cell transplant recipients: a population-based prospective study.

    Science.gov (United States)

    van Veen, K E B; Brouwer, M C; van der Ende, A; van de Beek, D

    2016-11-01

    We performed a nationwide prospective cohort study on the epidemiology and clinical features of community-acquired bacterial meningitis. Patients with a medical history of autologous or allogeneic hematopoietic stem cell transplantation (HSCT) were identified from the cohort performed from March 2006 to October 2014. Fourteen of 1449 episodes (1.0%) of bacterial meningitis occurred in patients with a history of HSCT. The incidence of bacterial meningitis in HSCT recipients was 40.4 per 100 000 patients per year (95% confidence interval (CI) 23.9-62.2), which is 30-fold (95% CI 18-51; Pmeningitis were infected with a serotype included in the 23-valent pneumococcal polysaccharide vaccine, of whom four developed meningitis despite vaccination. In conclusion, HSCT recipients have a substantially increased risk compared with the general population of acquiring bacterial meningitis, which is mostly due to S. pneumoniae, and disease is associated with high mortality and morbidity. Vaccination is important to prevent disease although vaccine failures did occur.

  3. Imaging of Bacterial and Fungal Cells Using Fluorescent Carbon Dots Prepared from Carica papaya Juice.

    Science.gov (United States)

    Kasibabu, Betha Saineelima B; D'souza, Stephanie L; Jha, Sanjay; Kailasa, Suresh Kumar

    2015-07-01

    In this paper, we have described a simple hydrothermal method for preparation of fluorescent carbon dots (C-dots) using Carica papaya juice as a precursor. The synthesized C-dots show emission peak at 461 nm with a quantum yield of 7.0 %. The biocompatible nature of C-dots was confirmed by a cytotoxicity assay on E. coli. The C-dots were used as fluorescent probes for imaging of bacterial (Bacillus subtilis) and fungal (Aspergillus aculeatus) cells and emitted green and red colors under different excitation wavelengths, which indicates that the C-dots can be used as a promising material for cell imaging.

  4. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions.

    Directory of Open Access Journals (Sweden)

    Sandra Schwarz

    2010-08-01

    Full Text Available Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs of Burkholderia thailandensis (B. thai in eukaryotic and bacterial cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ΔT6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions. From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas fluorescens and Serratia proteamaculans-leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly displaced in mixed biofilms with P. putida, whereas wild-type cells persisted and overran the competitor. Our data show that T6SSs within a single organism can have distinct functions in eukaryotic versus bacterial cell interactions. These systems are likely to be a decisive factor in the survival of bacterial cells of one species in intimate association with those of another, such as in polymicrobial communities present both in the environment and in many infections.

  5. Bacterial Biofilm Infection Detected in Breast Implant-Associated Anaplastic Large-Cell Lymphoma.

    Science.gov (United States)

    Hu, Honghua; Johani, Khalid; Almatroudi, Ahmad; Vickery, Karen; Van Natta, Bruce; Kadin, Marshall E; Brody, Garry; Clemens, Mark; Cheah, Chan Yoon; Lade, Stephen; Joshi, Preeti Avinash; Prince, H Miles; Deva, Anand K

    2016-06-01

    A recent association between breast implants and the development of anaplastic large-cell lymphoma (ALCL) has been observed. The purpose of this study was to identify whether bacterial biofilm is present in breast implant-associated ALCL and, if so, to compare the bacterial microbiome to nontumor capsule samples from breast implants with contracture. Twenty-six breast implant-associated ALCL samples were analyzed for the presence of biofilm by real-time quantitative polymerase chain reaction, next-generation sequencing, fluorescent in situ hybridization, and scanning electron microscopy, and compared to 62 nontumor capsule specimens. Both the breast implant-associated ALCL and nontumor capsule samples yielded high mean numbers of bacteria (breast implant-associated ALCL, 4.7 × 10 cells/mg of tissue; capsule, 4.9 × 10 cells/mg of tissue). Analysis of the microbiome in breast implant-associated ALCL specimens showed significant differences with species identified in nontumor capsule specimens. There was a significantly greater proportion of Ralstonia spp. present in ALCL specimens compared with nontumor capsule specimens (p capsule specimens compared with breast implant-associated ALCL specimens (p < 0.001). Bacterial biofilm was visualized both on scanning electron microscopy and fluorescent in situ hybridization. This novel finding of bacterial biofilm and a distinct microbiome in breast implant-associated ALCL samples points to a possible infectious contributing cause. Breast implants are widely used in both reconstructive and aesthetic surgery, and strategies to reduce their contamination should be more widely studied and practiced. Risk, V.

  6. Structural Insights into Protein-Protein Interactions Involved in Bacterial Cell Wall Biogenesis

    Directory of Open Access Journals (Sweden)

    Federica Laddomada

    2016-04-01

    Full Text Available The bacterial cell wall is essential for survival, and proteins that participate in its biosynthesis have been the targets of antibiotic development efforts for decades. The biosynthesis of its main component, the peptidoglycan, involves the coordinated action of proteins that are involved in multi-member complexes which are essential for cell division (the “divisome” and/or cell wall elongation (the “elongasome”, in the case of rod-shaped cells. Our knowledge regarding these interactions has greatly benefitted from the visualization of different aspects of the bacterial cell wall and its cytoskeleton by cryoelectron microscopy and tomography, as well as genetic and biochemical screens that have complemented information from high resolution crystal structures of protein complexes involved in divisome or elongasome formation. This review summarizes structural and functional aspects of protein complexes involved in the cytoplasmic and membrane-related steps of peptidoglycan biosynthesis, with a particular focus on protein-protein interactions whereby disruption could lead to the development of novel antibacterial strategies.

  7. Physical impaction injury effects on bacterial cells during spread plating influenced by cell characteristics of the organisms.

    Science.gov (United States)

    Thomas, P; Mujawar, M M; Sekhar, A C; Upreti, R

    2014-04-01

    To understand the factors that contribute to the variations in colony-forming units (CFU) in different bacteria during spread plating. Employing a mix culture of vegetative cells of ten organisms varying in cell characteristics (Gram reaction, cell shape and cell size), spread plating to the extent of just drying the agar surface (50-60 s) was tested in comparison with the alternate spotting-and-tilt-spreading (SATS) approach where 100 μl inoculum was distributed by mere tilting of plate after spotting as 20-25 microdrops. The former imparted a significant reduction in CFU by 20% over the spreader-independent SATS approach. Extending the testing to single organisms, Gram-negative proteobacteria with relatively larger cells (Escherichia, Enterobacter, Agrobacterium, Ralstonia, Pantoea, Pseudomonas and Sphingomonas spp.) showed significant CFU reduction with spread plating except for slow-growing Methylobacterium sp., while those with small rods (Xenophilus sp.) and cocci (Acinetobacter sp.) were less affected. Among Gram-positive nonspore formers, Staphylococcus epidermidis showed significant CFU reduction while Staphylococcus haemolyticus and actinobacteria (Microbacterium, Cellulosimicrobium and Brachybacterium spp.) with small rods/cocci were unaffected. Vegetative cells of Bacillus pumilus and B. subtilis were generally unaffected while others with larger rods (B. thuringiensis, Brevibacillus, Lysinibacillus and Paenibacillus spp.) were significantly affected. A simulated plating study coupled with live-dead bacterial staining endorsed the chances of cell disruption with spreader impaction in afflicted organisms. Significant reduction in CFU could occur during spread plating due to physical impaction injury to bacterial cells depending on the spreader usage and the variable effects on different organisms are determined by Gram reaction, cell size and cell shape. The inoculum spreader could impart physical disruption of vegetative cells against a hard surface

  8. Bacterial Signaling Nucleotides Inhibit Yeast Cell Growth by Impacting Mitochondrial and Other Specifically Eukaryotic Functions

    Directory of Open Access Journals (Sweden)

    Andy Hesketh

    2017-07-01

    Full Text Available We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP, cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection.

  9. Lysis of bacterial cells in the process of bacteriophage release – canonical and newly discovered mechanisms

    Directory of Open Access Journals (Sweden)

    Wioleta M. Woźnica

    2015-01-01

    Full Text Available The release of phage progeny from an infected bacterium is necessary for the spread of infection. Only helical phages are secreted from a cell without causing its destruction. The release of remaining phages is correlated with bacterial lysis and death. Thus, the understanding of phage lytic functions is crucial for their use in the fight with bacterial pathogens. Bacteriophages with small RNA or DNA genomes encode single proteins which are called amurins and cause lysis by the inhibition of cell wall synthesis. Bacteriophages of double-stranded DNA genomes, which dominate in the environment, encode enzymes that are called endolysins and contribute to lysis by the cleavage of cell wall peptydoglycan. Endolysins that do not contain signal sequences cannot pass the cytoplasmic membrane by themselves. Their access to peptidoglycan is provided by membrane proteins – holins, which can form in the membrane large pores, that are called “holes”. Some endolysins do not require holins for their transport, owing to the presence of the so called SAR sequence at their N-terminus. It enables their transport through the membrane by the bacterial sec system. However, it is not cleaved off, and thus these endolysins remain trapped in the membrane in an inactive form. Their release, which is correlated with the activation, occurs as a result of membrane depolarization and depends on proteins that are called pinholins. Pinholins form in membrane pores that are too small for the passage of endolysins but sufficient for membrane depolarization. Proteins that are called antiholins regulate the timing of lysis, through the blockage of holins action until the end of phage morphogenesis. Additionally, newly identified lytic proteins, spanins, participate in the release of progeny phages from Gram-negative bacteria cells. They cause the destruction of outer cell membrane by its spanning with the cytoplasmic membrane. This is possible after the endolysin

  10. The prototypical proton-coupled oligopeptide transporter YdgR from Escherichia coli facilitates chloramphenicol uptake into bacterial cells

    DEFF Research Database (Denmark)

    Prabhala, Bala K; Aduri, Nanda G; Sharma, Neha

    2018-01-01

    Chloramphenicol (Cam) is a broad-spectrum antibiotic used to combat bacterial infections in humans and animals. Cam export from bacterial cells is one of the mechanisms by which pathogens resist Cam's antibacterial effects, and several different proteins are known to facilitate this process....... However, to date no report exists on any specific transport protein that facilitates Cam uptake. The proton-coupled oligopeptide transporter (POT) YdgR from Escherichia coli is a prototypical member of the POT family, functioning in proton-coupled uptake of di- and tripeptides. By following bacterial...... suggested a binding mode that resembles that of Cam binding to the multidrug resistance transporter MdfA. To our knowledge, this is the first report of Cam uptake into bacterial cells mediated by a specific transporter protein. Our findings suggest a specific bacterial transporter for drug uptake that might...

  11. Inflammasome-mediated cell death in response to bacterial pathogens that access the host cell cytosol: lessons from Legionella pneumophila

    Directory of Open Access Journals (Sweden)

    Cierra Nichole Casson

    2013-12-01

    Full Text Available Cell death can be critical for host defense against intracellular pathogens because it eliminates a crucial replicative niche, and pro-inflammatory cell death can alert neighboring cells to the presence of pathogenic organisms and enhance downstream immune responses. Pyroptosis is a pro-inflammatory form of cell death triggered by the inflammasome, a multi-protein complex that assembles in the cytosol to activate caspase-1. Inflammasome activation by pathogens hinges upon violation of the host cell cytosol by activities such as the use of pore-forming toxins, the use of specialized secretion systems, or the cytosolic presence of the pathogen itself. Recently, a non-canonical inflammasome has been described that activates caspase-11 and also leads to pro-inflammatory cell death. Caspase-11 is activated rapidly and robustly in response to violation of the cytosol by bacterial pathogens as well. In this mini-review, we describe the canonical and non-canonical inflammasome pathways that are critical for host defense against a model intracellular bacterial pathogen that accesses the host cytosol—Legionella pneumophila.

  12. Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization.

    Science.gov (United States)

    Ursell, Tristan S; Nguyen, Jeffrey; Monds, Russell D; Colavin, Alexandre; Billings, Gabriel; Ouzounov, Nikolay; Gitai, Zemer; Shaevitz, Joshua W; Huang, Kerwyn Casey

    2014-03-18

    Cells typically maintain characteristic shapes, but the mechanisms of self-organization for robust morphological maintenance remain unclear in most systems. Precise regulation of rod-like shape in Escherichia coli cells requires the MreB actin-like cytoskeleton, but the mechanism by which MreB maintains rod-like shape is unknown. Here, we use time-lapse and 3D imaging coupled with computational analysis to map the growth, geometry, and cytoskeletal organization of single bacterial cells at subcellular resolution. Our results demonstrate that feedback between cell geometry and MreB localization maintains rod-like cell shape by targeting cell wall growth to regions of negative cell wall curvature. Pulse-chase labeling indicates that growth is heterogeneous and correlates spatially and temporally with MreB localization, whereas MreB inhibition results in more homogeneous growth, including growth in polar regions previously thought to be inert. Biophysical simulations establish that curvature feedback on the localization of cell wall growth is an effective mechanism for cell straightening and suggest that surface deformations caused by cell wall insertion could direct circumferential motion of MreB. Our work shows that MreB orchestrates persistent, heterogeneous growth at the subcellular scale, enabling robust, uniform growth at the cellular scale without requiring global organization.

  13. Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

    Directory of Open Access Journals (Sweden)

    Elizabeth Muir

    Full Text Available There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location.To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate.Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed

  14. The periplasmic enzyme, AnsB, of Shigella flexneri modulates bacterial adherence to host epithelial cells.

    Directory of Open Access Journals (Sweden)

    Divya T George

    Full Text Available S. flexneri strains, most frequently linked with endemic outbreaks of shigellosis, invade the colonic and rectal epithelium of their host and cause severe tissue damage. Here we have attempted to elucidate the contribution of the periplasmic enzyme, L-asparaginase (AnsB to the pathogenesis of S. flexneri. Using a reverse genetic approach we found that ansB mutants showed reduced adherence to epithelial cells in vitro and attenuation in two in vivo models of shigellosis, the Caenorhabditis elegans and the murine pulmonary model. To investigate how AnsB affects bacterial adherence, we compared the proteomes of the ansB mutant with its wild type parental strain using two dimensional differential in-gel electrophoresis and identified the outer membrane protein, OmpA as up-regulated in ansB mutant cells. Bacterial OmpA, is a prominent outer membrane protein whose activity has been found to be required for bacterial pathogenesis. Overexpression of OmpA in wild type S. flexneri serotype 3b resulted in decreasing the adherence of this virulent strain, suggesting that the up-regulation of OmpA in ansB mutants contributes to the reduced adherence of this mutant strain. The data presented here is the first report that links the metabolic enzyme AnsB to S. flexneri pathogenesis.

  15. Analysis of bone marrow stromal cell transferred bacterial {beta}-galactosidase gene by PIXE

    Energy Technology Data Exchange (ETDEWEB)

    Kumakawa, Toshiro [Tokyo Metropolitan Geriatric Hospital, Tokyo (Japan). Dept. of Blood Transfusion and Hematology; Hibino, Hitoshi; Tani, Kenzaburo; Asano, Shigetaka; Futatugawa, Shouji; Sera, Kouichiro

    1997-12-31

    PIXE, Particle Induced X-ray Emission, is a powerful, multi-elemental analysis method which has many distinguishing features and has been used in varies research fields. Recently the method of applying baby cyclotrons for nuclear medicine to PIXE has been developed. This enables us to study biomedical phenomena from the physical point of view. Mouse bone marrow stromal cells were transferred bacterial {beta}-galactosidase gene (LacZ gene) by murine retroviral vectors. Analysis of the bone marrow stromal cells with the LacZ gene by PIXE revealed remarkable changes of intracellular trace elements compared with the normal control cells. These results indicate that gene transfer by retroviral vectors may bring about a dynamic change of intracellular circumstances of the target cell. (author)

  16. Computer models of bacterial cells: from generalized coarsegrained to genome-specific modular models

    International Nuclear Information System (INIS)

    Nikolaev, Evgeni V; Atlas, Jordan C; Shuler, Michael L

    2006-01-01

    We discuss a modular modelling framework to rapidly develop mathematical models of bacterial cells that would explicitly link genomic details to cell physiology and population response. An initial step in this approach is the development of a coarse-grained model, describing pseudo-chemical interactions between lumped species. A hybrid model of interest can then be constructed by embedding genome-specific detail for a particular cellular subsystem (e.g. central metabolism), called here a module, into the coarse-grained model. Specifically, a new strategy for sensitivity analysis of the cell division limit cycle is introduced to identify which pseudo-molecular processes should be delumped to implement a particular biological function in a growing cell (e.g. ethanol overproduction or pathogen viability). To illustrate the modeling principles and highlight computational challenges, the Cornell coarsegrained model of Escherichia coli B/r-A is used to benchmark the proposed framework

  17. Subversion of the B-cell compartment during parasitic, bacterial, and viral infections.

    Science.gov (United States)

    Borhis, Gwenoline; Richard, Yolande

    2015-03-26

    Recent studies on HIV infection have identified new human B-cell subsets with a potentially important impact on anti-viral immunity. Current work highlights the occurrence of similar B-cell alterations in other viral, bacterial, and parasitic infections, suggesting that common strategies have been developed by pathogens to counteract protective immunity. For this review, we have selected key examples of human infections for which B-cell alterations have been described, to highlight the similarities and differences in the immune responses to a variety of pathogens. We believe that further comparisons between these models will lead to critical progress in the understanding of B-cell mechanisms and will open new target avenues for therapeutic interventions.

  18. Formulation of bacterial consortium as whole cell biocatalyst for degradation of oil compounds

    Science.gov (United States)

    Yetti, Elvi; A'la, Amalia; Luthfiyah, Nailul; Wijaya, Hans; Thontowi, Ahmad; Yopi

    2017-11-01

    In this research, weaim to investigateformulation of bacterial consortium as whole cell biocatalyst for degradation of oil compounds. We constructed microbial consortium from 4 (four) selected marine oil bacteria to become 15 (twelve) combination culture. Those bacteria were from collection of Laboratory of Biocatalyst and Fermentation, Research Center for Biotechnology, Indonesian Institutes of Sciences and designated as Labrenzia sp. MBTDCMFRIMab26, Labrenzia aggregata strasin HQB397, Novosphingobium pentaromativorans strain PQ-3 16S, and Novosphingobium pentaromativorans strain US6-1. The mixture or bacteria consortia, denoted as F1, F2, …F15 consisted of 1, 2, 3 and 4 bacterial strains, respectively. The strains were selected based on the criteria that they were able to display good growth in crude oil containing media. Five bacterialformulationsshowed good potentialas candidates for microbial consortium. We will optimize these consortium with carrier matrix choosed from biomass materials and also carry out oil content analysis.

  19. Modeling quorum sensing trade-offs between bacterial cell density and system extension from open boundaries

    Science.gov (United States)

    Marenda, Mattia; Zanardo, Marina; Trovato, Antonio; Seno, Flavio; Squartini, Andrea

    2016-12-01

    Bacterial communities undergo collective behavioural switches upon producing and sensing diffusible signal molecules; a mechanism referred to as Quorum Sensing (QS). Exemplarily, biofilm organic matrices are built concertedly by bacteria in several environments. QS scope in bacterial ecology has been debated for over 20 years. Different perspectives counterpose the role of density reporter for populations to that of local environment diffusivity probe for individual cells. Here we devise a model system where tubes of different heights contain matrix-embedded producers and sensors. These tubes allow non-limiting signal diffusion from one open end, thereby showing that population spatial extension away from an open boundary can be a main critical factor in QS. Experimental data, successfully recapitulated by a comprehensive mathematical model, demonstrate how tube height can overtake the role of producer density in triggering sensor activation. The biotic degradation of the signal is found to play a major role and to be species-specific and entirely feedback-independent.

  20. Total bacterial count and somatic cell count in refrigerated raw milk stored in communal tanks

    Directory of Open Access Journals (Sweden)

    Edmar da Costa Alves

    2014-09-01

    Full Text Available The current industry demand for dairy products with extended shelf life has resulted in new challenges for milk quality maintenance. The processing of milk with high bacterial counts compromises the quality and performance of industrial products. The study aimed to evaluate the total bacteria counts (TBC and somatic cell count (SCC in 768 samples of refrigerated raw milk, from 32 communal tanks. Samples were collected in the first quarter of 2010, 2011, 2012 and 2013 and analyzed by the Laboratory of Milk Quality - LQL. Results showed that 62.5%, 37.5%, 15.6% and 27.1% of the means for TBC in 2010, 2011, 2012 and 2013, respectively, were above the values established by legislation. However, we observed a significant reduction in the levels of total bacterial count (TBC in the studied periods. For somatic cell count, 100% of the means indicated values below 600.000 cells/mL, complying with the actual Brazilian legislation. The values found for the somatic cell count suggests the adoption of effective measures for the sanitary control of the herd. However, the results must be considered with caution as it highlights the need for quality improvements of the raw material until it achieves reliable results effectively.

  1. Assessment of synergistic antibacterial activity of combined biosurfactants revealed by bacterial cell envelop damage.

    Science.gov (United States)

    Sana, Santanu; Datta, Sriparna; Biswas, Dipa; Sengupta, Dipanjan

    2018-02-01

    Besides potential surface activity and some beneficial physical properties, biosurfactants express antibacterial activity. Bacterial cell membrane disrupting ability of rhamnolipid produced by Pseudomonas aeruginosa C2 and a lipopeptide type biosurfactant, BS15 produced by Bacillus stratosphericus A15 was examined against Staphylococcus aureus ATCC 25923 and Escherichia coli K8813. Broth dilution technique was followed to examine minimum inhibitory concentration (MIC) of both the biosurfactants. The combined effect of rhamnolipid and BS15 against S. aureus and E. coli showed synergistic activity by expressing fractional inhibitory concentration (FIC) index of 0.43 and 0.5. Survival curve of both the bacteria showed bactericidal activity after treating with biosurfactants at their MIC obtained from FIC index study as it killed >90% of initial population. The lesser value of MIC than minimum bactericidal concentration (MBC) of the biosurfactants also supported their bactericidal activity against both the bacteria. Membrane permeability against both the bacteria was supported by amplifying protein release, increasing of cell surface hydrophobicity, withholding capacity of crystal violet dye and leakage of intracellular materials. Finally cell membrane disruption was confirmed by scanning electron microscopy (SEM). All these experiments expressed synergism and effective bactericidal activity of the combination of rhamnolipid and BS15 by enhancing the bacterial cell membrane permeability. Such effect of the combination of rhamnolipid and BS15 could make them promising alternatives to traditional antibiotic in near future. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Effect of bacterial cell-free supernatants on infectivity of norovirus surrogates.

    Science.gov (United States)

    Shearer, Adrienne E H; Hoover, Dallas G; Kniel, Kalmia E

    2014-01-01

    Bacterial metabolic products were evaluated for inhibitory effects on viral propagation in cell culture. Cell-free supernatants (CFS) were prepared from growth of Enterococcus faecalis ATCC 19433, Pseudomonas fluorescens ATCC 13525, Escherichia coli 08, Staphylococcus epidermidis ATCC 12228, Bacillus subtilis 168, Bacillus coagulans 185A, B. coagulans 7050, Clostridium sporogenes PA3679, and a commercial probiotic mixture of Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium bifidum, Lactobacillus salivarius, and Streptococcus thermophilus in microbiological medium or milk. The inhibitory effects of CFS on the propagation of murine norovirus 1 and Tulane virus in RAW 264.7 and LLCMK2 cells, respectively, were evaluated in the continuous presence of CFS or after exposure of host cells to CFS. Slight inhibition of viral propagation was observed for murine norovirus and Tulane virus in the continuous presence of CFS of B. subtilis 168 and E. faecalis 19433, respectively. CFS cytotoxicity was also determined by microscopic examination. Virus persisted in the CFS that demonstrated cytotoxic effects, suggesting a lack of direct effect of CFS on virions. The viral propagation indicates a general lack of competitive inhibition by bacterial extracellular products and bears significance in understanding the persistence of virus in food and human systems shared by bacteria that are recognized for their colonization and competitive capabilities.

  3. Light scattering on PHA granules protects bacterial cells against the harmful effects of UV radiation.

    Science.gov (United States)

    Slaninova, Eva; Sedlacek, Petr; Mravec, Filip; Mullerova, Lucie; Samek, Ota; Koller, Martin; Hesko, Ondrej; Kucera, Dan; Marova, Ivana; Obruca, Stanislav

    2018-02-01

    Numerous prokaryotes accumulate polyhydroxyalkanoates (PHA) in the form of intracellular granules. The primary function of PHA is the storage of carbon and energy. Nevertheless, there are numerous reports that the presence of PHA granules in microbial cells enhances their stress resistance and fitness when exposed to various stress factors. In this work, we studied the protective mechanism of PHA granules against UV irradiation employing Cupriavidus necator as a model bacterial strain. The PHA-accumulating wild type strain showed substantially higher UV radiation resistance than the PHA non-accumulating mutant. Furthermore, the differences in UV-Vis radiation interactions with both cell types were studied using various spectroscopic approaches (turbidimetry, absorption spectroscopy, and nephelometry). Our results clearly demonstrate that intracellular PHA granules efficiently scatter UV radiation, which provides a substantial UV-protective effect for bacterial cells and, moreover, decreases the intracellular level of reactive oxygen species in UV-challenged cells. The protective properties of the PHA granules are enhanced by the fact that granules specifically bind to DNA, which in turn provides shield-like protection of DNA as the most UV-sensitive molecule. To conclude, the UV-protective action of PHA granules adds considerable value to their primary storage function, which can be beneficial in numerous environments.

  4. Effect of a small molecule Lipid II binder on bacterial cell wall stress

    Directory of Open Access Journals (Sweden)

    Malin J

    2017-02-01

    Full Text Available Jakob Malin,1,2 Amol C Shetty,3 Sean Daugherty,3 Erik PH de Leeuw,1,2 1Institute of Human Virology, 2Department of Biochemistry and Molecular Biology, 3Institute for Genome Sciences, University of Maryland Baltimore School of Medicine, Baltimore, MD, USA Abstract: We have recently identified small molecule compounds that act as binders of Lipid II, an essential precursor of bacterial cell wall biosynthesis. Lipid II comprised a hydrophilic head group that includes a peptidoglycan subunit composed of N-acetylglucosamine (GlcNAc and N-acetylmuramic acid (MurNAc coupled to a short pentapeptide moiety. This headgroup is coupled to a long bactoprenol chain via a pyrophosphate group. Here, we report on the cell wall activity relationship of dimethyl-3-methyl(phenylamino-ethenylcyclohexylidene-propenyl-3-ethyl-1,3-benzothiazolium iodide (compound 5107930 obtained by functional and genetic analyses. Our results indicate that compounds bind to Lipid II and cause specific upregulation of the vancomycin-resistance associated gene vraX. vraX is implicated in the cell wall stress stimulon that confers glycopeptide resistance. Our small molecule Lipid II inhibitor retained activity against strains of Staphylococcus aureus mutated in genes encoding the cell wall stress stimulon. This suggests the feasibility of developing this new scaffold as a therapeutic agent in view of increasing glycopeptide resistance. Keywords: defensin, Lipid II, antibiotics, bacterial membrane, vancomycin

  5. Development of Phage Lysin LysA2 for Use in Improved Purity Assays for Live Biotherapeutic Products

    Directory of Open Access Journals (Sweden)

    Sheila M. Dreher-Lesnick

    2015-12-01

    Full Text Available Live biotherapeutic products (LBPs, commonly referred to as probiotics, are typically preparations of live bacteria, such as Lactobacillus and Bifidobacterium species that are considered normal human commensals. Popular interest in probiotics has been increasing with general health benefits being attributed to their consumption, but there is also growing interest in evaluating such products for treatment of specific diseases. While over-the-counter probiotics are generally viewed as very safe, at least in healthy individuals, it must be remembered that clinical studies to assess these products may be done in individuals whose defenses are compromised, such as through a disease process, immunosuppressive clinical treatment, or an immature or aging immune system. One of the major safety criteria for LBPs used in clinical studies is microbial purity, i.e., the absence of extraneous, undesirable microorganisms. The main goal of this project is to develop recombinant phage lysins as reagents for improved purity assays for LBPs. Phage lysins are hydrolytic enzymes containing a cell binding domain that provides specificity and a catalytic domain responsible for lysis and killing. Our approach is to use recombinant phage lysins to selectively kill target product bacteria, which when used for purity assays will allow for outgrowth of potential contaminants under non-selective conditions, thus allowing an unbiased assessment of the presence of contaminants. To develop our approach, we used LysA2, a phage lysin with reported activity against a broad range of Lactobacillus species. We report the lytic profile of a non-tagged recombinant LysA2 against Lactobacillus strains in our collection. We also present a proof-of-concept experiment, showing that addition of partially purified LysA2 to a culture of Lactobacillus jensenii (L. jensenii spiked with low numbers of Escherichia coli (E. coli or Staphylococcus aureus (S. aureus effectively eliminates or knocks

  6. Real-time Bacterial Detection by Single Cell Based Sensors UsingSynchrotron FTIR Spectromicroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Veiseh, Mandana; Veiseh, Omid; Martin, Michael C.; Bertozzi,Carolyn; Zhang, Miqin

    2005-08-10

    Microarrays of single macrophage cell based sensors weredeveloped and demonstrated for real time bacterium detection bysynchrotron FTIR microscopy. The cells were patterned on gold-SiO2substrates via a surface engineering technique by which the goldelectrodes were immobilized with fibronectin to mediate cell adhesion andthe silicon oxide background were passivated with PEG to resist proteinadsorption and cell adhesion. Cellular morphology and IR spectra ofsingle, double, and triple cells on gold electrodes exposed tolipopolysaccharide (LPS) of different concentrations were compared toreveal the detection capabilities of these biosensors. The single-cellbased sensors were found to generate the most significant IR wave numbervariation and thus provide the highest detection sensitivity. Changes inmorphology and IR spectrum for single cells exposed to LPS were found tobe time- and concentration-dependent and correlated with each other verywell. FTIR spectra from single cell arrays of gold electrodes withsurface area of 25 mu-m2, 100 mu-m2, and 400 mu-m2 were acquired usingboth synchrotron and conventional FTIR spectromicroscopes to study thesensitivity of detection. The results indicated that the developedsingle-cell platform can be used with conventional FTIRspectromicroscopy. This technique provides real-time, label-free, andrapid bacterial detection, and may allow for statistic and highthroughput analyses, and portability.

  7. The role of T cell subsets and cytokines in the regulation of intracellular bacterial infection

    Directory of Open Access Journals (Sweden)

    Oliveira S.C.

    1998-01-01

    Full Text Available Cellular immune responses are a critical part of the host's defense against intracellular bacterial infections. Immunity to Brucella abortus crucially depends on antigen-specific T cell-mediated activation of macrophages, which are the major effectors of cell-mediated killing of this organism. T lymphocytes that proliferate in response to B. abortus were characterized for phenotype and cytokine activity. Human, murine, and bovine T lymphocytes exhibited a type 1 cytokine profile, suggesting an analogous immune response in these different hosts. In vivo protection afforded by a particular cell type is dependent on the antigen presented and the mechanism of antigen presentation. Studies using MHC class I and class II knockout mice infected with B. abortus have demonstrated that protective immunity to brucellosis is especially dependent on CD8+ T cells. To target MHC class I presentation we transfected ex vivo a murine macrophage cell line with B. abortus genes and adoptively transferred them to BALB/c mice. These transgenic macrophage clones induced partial protection in mice against experimental brucellosis. Knowing the cells required for protection, vaccines can be designed to activate the protective T cell subset. Lastly, as a new strategy for priming a specific class I-restricted T cell response in vivo, we used genetic immunization by particle bombardment-mediated gene transfer

  8. Nanoscale imaging of the growth and division of bacterial cells on planar substrates with the atomic force microscope

    Energy Technology Data Exchange (ETDEWEB)

    Van Der Hofstadt, M. [Institut de Bioenginyeria de Catalunya (IBEC), C/ Baldiri i Reixac 11-15, 08028 Barcelona (Spain); Hüttener, M.; Juárez, A. [Institut de Bioenginyeria de Catalunya (IBEC), C/ Baldiri i Reixac 11-15, 08028 Barcelona (Spain); Departament de Microbiologia, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona (Spain); Gomila, G., E-mail: ggomila@ibecbarcelona.eu [Institut de Bioenginyeria de Catalunya (IBEC), C/ Baldiri i Reixac 11-15, 08028 Barcelona (Spain); Departament d' Electronica, Universitat de Barcelona, C/ Marti i Franqués 1, 08028 Barcelona (Spain)

    2015-07-15

    With the use of the atomic force microscope (AFM), the Nanomicrobiology field has advanced drastically. Due to the complexity of imaging living bacterial processes in their natural growing environments, improvements have come to a standstill. Here we show the in situ nanoscale imaging of the growth and division of single bacterial cells on planar substrates with the atomic force microscope. To achieve this, we minimized the lateral shear forces responsible for the detachment of weakly adsorbed bacteria on planar substrates with the use of the so called dynamic jumping mode with very soft cantilever probes. With this approach, gentle imaging conditions can be maintained for long periods of time, enabling the continuous imaging of the bacterial cell growth and division, even on planar substrates. Present results offer the possibility to observe living processes of untrapped bacteria weakly attached to planar substrates. - Highlights: • Gelatine coatings used to weakly attach bacterial cells onto planar substrates. • Use of the dynamic jumping mode as a non-perturbing bacterial imaging mode. • Nanoscale resolution imaging of unperturbed single living bacterial cells. • Growth and division of single bacteria cells on planar substrates observed.

  9. The use of a cloned bacterial gene to study mutation in mammalian cells

    International Nuclear Information System (INIS)

    Thacker, J.; Debenham, P.G.; Stretch, A.; Webb, M.B.T.

    1983-01-01

    The recombinant DNA molecule pSV2-gpt, which contains the bacterial gene coding for xanthine-guanine phosphoribosyl transferase (XGPRT) activity, was introduced into a hamster cell line lacking the equivalent mammalian enzyme (HGPRT). Hamster cell sublines were found with stable expression of XGPRT activity and were used to study mutation of the integrated pSV2-gpt DNA sequence. Mutants were selected by their resistance to 6-thioguanine (TG) under optimal conditions which were found to be very similar to those for selection of HGPRT-deficient mutants of mammalian cells. The frequency of XGPRT-deficient mutants was increased by treatment with X-rays, ethyl methanesulphonate and ethyl nitrosourea. X-Ray induction of mutants increased approximately linearly with dose up to about 500 rad, but the frequency of mutants per rad was very much higher than that usually found for 'native' mammalian genes. (orig./AJ)

  10. Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization

    DEFF Research Database (Denmark)

    Schou, Kirstine Klitgaard; Mølbak, Lars; Jensen, Tim Kåre

    2005-01-01

    Direct cultivation-independent sequence retrieval of unidentified bacteria from histological tissue sections has been limited by the difficulty of selectively isolating specific bacteria from a complex environment. Here, a new DNA isolation approach is presented for prokaryotic cells....... By this method, a potentially pathogenic strain of the genus Brachyspira from formalin-fixed human colonic biopsies were visualized by fluorescence in situ hybridization (FISH) with a 16S rRNA-targeting oligonucleotide probe, followed by laser capture microdissection (LCM) of the targeted cells. Direct 16S r......RNA gene PCR was performed from the dissected microcolonies, and the subsequent DNA sequence analysis identified the dissected bacterial cells as belonging to the Brachyspira aalborgi cluster 1. The advantage of this technique is the ability to combine the histological recognition of the specific bacteria...

  11. Effect of media components on cell growth and bacterial cellulose production from Acetobacter aceti MTCC 2623.

    Science.gov (United States)

    Dayal, Manmeet Singh; Goswami, Navendu; Sahai, Anshuman; Jain, Vibhor; Mathur, Garima; Mathur, Ashwani

    2013-04-15

    Acetobacter aceti MTCC 2623 was studied as an alternative microbial source for bacterial cellulose (BC) production. Effect of media components on cell growth rate, BC production and cellulose characteristics were studied. FTIR results showed significant variations in cellulose characteristics produced by A. aceti in different media. Results have shown the role of fermentation time on crystallinity ratio of BC in different media. Further, effect of six different media components on cell growth and BC production was studied using fractional factorial design. Citric acid was found to be the most significant media component for cell growth rate (95% confidence level, R(2)=0.95). However, direct role of these parameters on cellulose production was not established (p-value>0.05). Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Role of pigmentation in protecting bacterial cells against irradiation generated by accelerated charged particles

    International Nuclear Information System (INIS)

    Tiwary, Bhupendra Nath; Das, Reena

    2013-01-01

    Beams of high-energy particles are useful for both fundamental and applied research in the sciences, and also in many technical and industrial fields unrelated to fundamental research. It has been estimated that there are approximately 26,000 accelerators world. Of these, only about 1% are research machines with energies above 1 GeV, while about 44% are for radiotherapy, 41% for ion implantation, 9% for industrial processing and research, and 4% for biomedical and other low-energy research. One aspect of these radiations can be studied for examining their effect in altering the viability of bacterial cells. The radiations generated by the simple technology of a single static high voltage to accelerate charged particles are known to produce reactive oxygen intermediates such as hydrogen peroxide or superoxide anions and target several cellular components of bacterial cells including the DNA. As a result of this interaction with the DNA the phosphodiester backbone of the DNA may break leading to single or double strand fission. Endogenous pigments, such as carotenoids and melanins, might provide a selective advantage to these microorganisms by photoprotection or shielding from UV radiation, including the UV-C and full UV-B range. The pigment, as an antioxidant scavenges reactive oxygen species generated by UV-A radiation and protect various microorganisms against oxidative damage caused by UV or ionizing radiation by scavenging free radicals. Environmental UV radiation is polychromatic and comprises the full spectrum of UV-A and UV-B radiation at wavelengths of λ > 290 nm. Accelerators, solar simulators and natural insulation can also prove to be a better alternate for understanding the responses of bacterial cells to the terrestrial UV radiation climate. (author)

  13. Phylogenetic and metagenomic analyses of substrate-dependent bacterial temporal dynamics in microbial fuel cells.

    Directory of Open Access Journals (Sweden)

    Husen Zhang

    Full Text Available Understanding the microbial community structure and genetic potential of anode biofilms is key to improve extracellular electron transfers in microbial fuel cells. We investigated effect of substrate and temporal dynamics of anodic biofilm communities using phylogenetic and metagenomic approaches in parallel with electrochemical characterizations. The startup non-steady state anodic bacterial structures were compared for a simple substrate, acetate, and for a complex substrate, landfill leachate, using a single-chamber air-cathode microbial fuel cell. Principal coordinate analysis showed that distinct community structures were formed with each substrate type. The bacterial diversity measured as Shannon index decreased with time in acetate cycles, and was restored with the introduction of leachate. The change of diversity was accompanied by an opposite trend in the relative abundance of Geobacter-affiliated phylotypes, which were acclimated to over 40% of total Bacteria at the end of acetate-fed conditions then declined in the leachate cycles. The transition from acetate to leachate caused a decrease in output power density from 243±13 mW/m2 to 140±11 mW/m2, accompanied by a decrease in Coulombic electron recovery from 18±3% to 9±3%. The leachate cycles selected protein-degrading phylotypes within phylum Synergistetes. Metagenomic shotgun sequencing showed that leachate-fed communities had higher cell motility genes including bacterial chemotaxis and flagellar assembly, and increased gene abundance related to metal resistance, antibiotic resistance, and quorum sensing. These differentially represented genes suggested an altered anodic biofilm community in response to additional substrates and stress from the complex landfill leachate.

  14. Bacterial host and reporter gene optimization for genetically encoded whole cell biosensors.

    Science.gov (United States)

    Brutesco, Catherine; Prévéral, Sandra; Escoffier, Camille; Descamps, Elodie C T; Prudent, Elsa; Cayron, Julien; Dumas, Louis; Ricquebourg, Manon; Adryanczyk-Perrier, Géraldine; de Groot, Arjan; Garcia, Daniel; Rodrigue, Agnès; Pignol, David; Ginet, Nicolas

    2017-01-01

    Whole-cell biosensors based on reporter genes allow detection of toxic metals in water with high selectivity and sensitivity under laboratory conditions; nevertheless, their transfer to a commercial inline water analyzer requires specific adaptation and optimization to field conditions as well as economical considerations. We focused here on both the influence of the bacterial host and the choice of the reporter gene by following the responses of global toxicity biosensors based on constitutive bacterial promoters as well as arsenite biosensors based on the arsenite-inducible P ars promoter. We observed important variations of the bioluminescence emission levels in five different Escherichia coli strains harboring two different lux-based biosensors, suggesting that the best host strain has to be empirically selected for each new biosensor under construction. We also investigated the bioluminescence reporter gene system transferred into Deinococcus deserti, an environmental, desiccation- and radiation-tolerant bacterium that would reduce the manufacturing costs of bacterial biosensors for commercial water analyzers and open the field of biodetection in radioactive environments. We thus successfully obtained a cell survival biosensor and a metal biosensor able to detect a concentration as low as 100 nM of arsenite in D. deserti. We demonstrated that the arsenite biosensor resisted desiccation and remained functional after 7 days stored in air-dried D. deserti cells. We also report here the use of a new near-infrared (NIR) fluorescent reporter candidate, a bacteriophytochrome from the magnetotactic bacterium Magnetospirillum magneticum AMB-1, which showed a NIR fluorescent signal that remained optimal despite increasing sample turbidity, while in similar conditions, a drastic loss of the lux-based biosensors signal was observed.

  15. Ion Channels Activated by Mechanical Forces in Bacterial and Eukaryotic Cells.

    Science.gov (United States)

    Sokabe, Masahiro; Sawada, Yasuyuki; Kobayashi, Takeshi

    2015-01-01

    Since the first discovery of mechanosensitive ion channel (MSC) in non-sensory cells in 1984, a variety of MSCs has been identified both in prokaryotic and eukaryotic cells. One of the central issues concerning MSCs is to understand the molecular and biophysical mechanisms of how mechanical forces activate/open MSCs. It has been well established that prokaryotic (mostly bacterial) MSCs are activated exclusively by membrane tension. Thus the problem to be solved with prokaryotic MSCs is the mechanisms how the MSC proteins receive tensile forces from the lipid bilayer and utilize them for channel opening. On the other hand, the activation of many eukaryotic MSCs crucially depends on tension in the actin cytoskeleton. By using the actin cytoskeleton as a force sensing antenna, eukaryotic MSCs have obtained sophisticated functions such as remote force sensing and force-direction sensing, which bacterial MSCs do not have. Actin cytoskeletons also give eukaryotic MSCs an interesting and important function called "active touch sensing", by which cells can sense rigidity of their substrates. The contractile actin cytoskeleton stress fiber (SF) anchors its each end to a focal adhesion (FA) and pulls the substrate to generate substrate-rigidity-dependent stresses in the FA. It has been found that those stresses are sensed by some Ca2+-permeable MSCs existing in the vicinity of FAs, thus the MSCs work as a substrate rigidity sensor that can transduce the rigidity into intracellular Ca2+ levels. This short review, roughly constituting of two parts, deals with molecular and biophysical mechanisms underlying the MSC activation process mostly based on our recent studies; (1) structure-function in bacterial MSCs activation at the atomic level, and (2) roles of actin cytoskeletons in the activation of eukaryotic MSCs.

  16. Comparative study of HOCl-inflicted damage to bacterial DNA ex vivo and within cells

    Science.gov (United States)

    Suquet, Christine; Warren, Jeffrey J.; Seth, Nimulrith; Hurst, James K.

    2009-01-01

    The prospects for using bacterial DNA as an intrinsic probe for HOCl and secondary oxidants/chlorinating agents associated with it has been evaluated using both in vitro and in vivo studies. Single-strand and double-strand breaks occurred in bare plasmid DNA that had been exposed to high levels of HOCl, although these reactions were very inefficient compared to polynucleotide chain cleavage caused by the OH•-generating reagent, peroxynitrite. Plasmid nicking was not increased when intact Escherichia coli were exposed to HOCl; rather, the amount of recoverable plasmid diminished in a dose-dependent manner. At concentration levels of HOCl exceeding lethal doses, genomic bacterial DNA underwent extensive fragmentation and the amount of precipitable DNA-protein complexes increased several-fold. The 5-chlorocytosine content of plasmid and genomic DNA isolated from HOCl-exposed E. coli was also slightly elevated above controls, as measured by mass spectrometry of the deaminated product, 5-chlorouracil. However, the yields were not dose-dependent over the bactericidal concentration range. Genomic DNA recovered from E. coli that had been subjected to phagocytosis by human neutrophils occasionally showed small increases in 5-chlorocytosine content when compared to analogous cellular reactions where myeloperoxidase activity was inhibited by azide ion. Overall, the amount of isolable 5-chlorouracil from the HOCl-exposed bacterial cells was far less than the damage manifested in polynucleotide bond cleavage and cross-linking. PMID:19850004

  17. Modification of N-glycosylation sites allows secretion of bacterial chondroitinase ABC from mammalian cells

    Science.gov (United States)

    Muir, Elizabeth M.; Fyfe, Ian; Gardiner, Sonya; Li, Li; Warren, Philippa; Fawcett, James W.; Keynes, Roger J.; Rogers, John H.

    2010-01-01

    Although many eukaryotic proteins have been secreted by transfected bacterial cells, little is known about how a bacterial protein is treated as it passes through the secretory pathway when expressed in a eukaryotic cell. The eukaryotic N-glycosylation system could interfere with folding and secretion of prokaryotic proteins whose sequence has not been adapted for glycosylation in structurally appropriate locations. Here we show that such interference does indeed occur for chondroitinase ABC from the bacterium Proteus vulgaris, and can be overcome by eliminating potential N-glycosylation sites. Chondroitinase ABC was heavily glycosylated when expressed in mammalian cells or in a mammalian translation system, and this process prevented secretion of functional enzyme. Directed mutagenesis of selected N-glycosylation sites allowed efficient secretion of active chondroitinase. As these proteoglycans are known to inhibit regeneration of axons in the mammalian central nervous system, the modified chondroitinase gene is a potential tool for gene therapy to promote neural regeneration, ultimately in human spinal cord injury. PMID:19900493

  18. Does penile tourniquet application alter bacterial adhesion to rat urethral cells: an in vitro study.

    Science.gov (United States)

    Boybeyi-Turer, Ozlem; Kacmaz, Birgul; Arat, Esra; Atasoy, Pınar; Kisa, Ucler; Gunal, Yasemin Dere; Aslan, Mustafa Kemal; Soyer, Tutku

    2018-04-01

    To investigate the effects of penile tourniquet (PT) application on bacterial adhesion to urothelium. Fifty-six rats were allocated into control group (CG), sham group (SG), PT group (PTG). No intervention was applied in CG. A 5mm-length urethral repair was performed in SG and PTG. In PTG, a 10-min duration of PT was applied during the procedure and the tissue oxygenation monitor was used to adjust the same degree of ischemia in all subjects. Samples were examined for wound healing parameters and tissue levels of inflammatory markers, eNOS, e-selectin, and ICAM-1antibodies. The adhesion of Escherichia coli to urothelium was investigated with in vitro adhesion assay. Inflammation was higher and wound healing was worse in SG than CG and in PTG in comparison to CG and SG (pcaused endothelial corruption and prevented cell proliferation in cell culture. The PT application does not improve wound healing and increases bacterial adhesion molecules in penile tissue. The in vitro assays showed that PT causes severe endothelial damage and inhibits endothelial cell proliferation. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Probing Induced Structural Changes in Biomimetic Bacterial Cell Membrane Interactions with Divalent Cations

    Energy Technology Data Exchange (ETDEWEB)

    Holt, Allison M [ORNL; Standaert, Robert F [ORNL; Jubb, Aaron M [ORNL; Katsaras, John [ORNL; Johs, Alexander [ORNL

    2017-01-01

    Biological membranes, formed primarily by the self-assembly of complex mixtures of phospholipids, provide a structured scaffold for compartmentalization and structural processes in living cells. The specific physical properties of phospholipid species present in a given membrane play a key role in mediating these processes. Phosphatidylethanolamine (PE), a zwitterionic lipid present in bacterial, yeast, and mammalian cell membranes, is exceptional. In addition to undergoing the standard lipid polymorphic transition between the gel and liquid-crystalline phase, it can also assume an unusual polymorphic state, the inverse hexagonal phase (HII). Divalent cations are among the factors that drive the formation of the HII phase, wherein the lipid molecules form stacked tubular structures by burying the hydrophilic head groups and exposing the hydrophobic tails to the bulk solvent. Most biological membranes contain a lipid species capable of forming the HII state suggesting that such lipid polymorphic structural states play an important role in structural biological processes such as membrane fusion. In this study, the interactions between Mg2+ and biomimetic bacterial cell membranes composed of PE and phosphatidylglycerol (PG) were probed using differential scanning calorimetry (DSC), small-angle x-ray scattering (SAXS), and fluorescence spectroscopy. The lipid phase transitions were examined at varying ratios of PE to PG and upon exposure to physiologically relevant concentrations of Mg2+. An understanding of these basic interactions enhances our understanding of membrane dynamics and how membrane-mediated structural changes may occur in vivo.

  20. Applications of whole-cell bacterial sensors in biotechnology and environmental science

    Energy Technology Data Exchange (ETDEWEB)

    Yagi, Kiyohito [Osaka Univ., Suita (Japan). Graduate School of Pharmaceutical Sciences

    2007-01-15

    Biosensors have major advantages over chemical or physical analyses with regard to specificity, sensitivity, and portability. Recently, many types of whole-cell bacterial biosensors have been developed using recombinant DNA technology. The bacteria are genetically engineered to respond to the presence of chemicals or physiological stresses by synthesizing a reporter protein, such as luciferase, {beta}-galactosidase, or green fluorescent protein. In addition to an overview of conventional biosensors, this minireview discusses a novel type of biosensor using a photosynthetic bacterium as the sensor strain and the crtA gene, which is responsible for carotenoid synthesis, as the reporter. Since bacteria possess a wide variety of stress-response mechanisms, including antioxidation, heat-shock responses, nutrient-starvation, and membrane-damage responses, DNA response elements for several stress-response proteins can be fused with various reporter genes to construct a versatile set of bacterial biosensors for a variety of analytes. Portable biosensors for on-site monitoring have been developed using a freeze-dried biosensing strain, and cell array biosensors have been designed for high-throughput analysis. Moreover, in the future, the use of single-cell biosensors will permit detailed analyses of samples. Signals from such sensors could be detected with digital imaging, epifluorescence microscopy, and/or flow cytometry. (orig.)

  1. Case Study: A Novel Bacterial Contamination in Cell Culture Production--Leptospira licerasiae.

    Science.gov (United States)

    Chen, Joseph; Bergevin, Jesse; Kiss, Robert; Walker, Gordon; Battistoni, Todd; Lufburrow, Patricia; Lam, Harry; Vinther, Anders

    2012-01-01

    Leptospira licerasiae, a novel bacterial contaminant found in Genentech cell culture manufacturing operations, poses a challenge to current microbial control strategies in upstream cell culture processes, as this microorganism is fully capable of passing through 0.1 μm sterilizing-grade filtration and is not detectable by standard microbiological methods described in major pharmaceutical compendia for microbial screening and quantification required for release of raw materials, in-process intermediates, and finished products in biopharmaceutical production. The root cause investigation was greatly aided by the genetic identification of the contaminant and subsequent confirmation by cultural method and real-time polymerase chain reaction assay from the affected product batches. The purpose of this case study is to share knowledge on the novel contaminant, L. licerasiae, and potential routes of contamination in the cell culture manufacturing environment from a series of investigations involving root cause analysis, impact assessments, risk assessment, and global corrective and preventative action, as well as to provide guidance on the detection and prevention of Leptospira contamination with the intent to aid the industry to continually improve microbial control strategies for the benefit of patients. Leptospira licerasiae, a novel bacterial contaminant found in cell culture manufacturing operations, poses a challenge to current microbial control strategies in upstream cell culture processes because this microorganism is capable of passing through 0.1 μm sterilizing-grade membrane filters and is not detectable by standard microbiological methods used in biopharmaceutical production. The root cause investigation was greatly aided by the genetic identification of the contaminant and subsequent confirmation by cultural method and real-time polymerase chain reaction assay from the affected product batches. The purpose of this case study is to share knowledge on the

  2. Modeling the cost and benefit of proteome regulation in a growing bacterial cell.

    Science.gov (United States)

    Sharma, Pooja; Pandey, Parth Pratim; Jain, Sanjay

    2018-04-16

    Escherichia coli cells differentially regulate the production of metabolic and ribosomal proteins in order to stay close to an optimal growth rate in different environments, and exhibit the bacterial growth laws as a consequence. We present a simple mathematical model of a growing-dividing cell in which an internal dynamical mechanism regulates the allocation of proteomic resources between different protein sectors. The model allows an endogenous determination of the growth rate of the cell as a function of cellular and environmental parameters, and reproduces the bacterial growth laws. We use the model and its variants to study the balance between the cost and benefit of regulation. A cost is incurred because cellular resources are diverted to produce the regulatory apparatus. We show that there is a window of environments or a 'niche' in which the unregulated cell has a higher fitness than the regulated cell. Outside this niche there is a large space of constant and time varying environments in which regulation is an advantage. A knowledge of the 'niche boundaries' allows one to gain an intuitive understanding of the class of environments in which regulation is an advantage for the organism and which would therefore favour the evolution of regulation. The model allows us to determine the 'niche boundaries' as a function of cellular parameters such as the size of the burden of the regulatory apparatus. This class of models may be useful in elucidating various tradeoffs in cells and in making in-silico predictions relevant for synthetic biology. © 2018 IOP Publishing Ltd.

  3. Probiotic Saccharomyces cerevisiae strains as biotherapeutic tools: is there room for improvement?

    Science.gov (United States)

    Palma, Mariana L; Zamith-Miranda, Daniel; Martins, Flaviano S; Bozza, Fernando A; Nimrichter, Leonardo; Montero-Lomeli, Mônica; Marques, Ernesto T A; Douradinha, Bruno

    2015-08-01

    The probiotic yeast Saccharomyces cerevisiae var boulardii is widely used as a low cost and efficient adjuvant against gastrointestinal tract disorders such as inflammatory bowel disease and treatment of several types of diarrhea, both in humans and animals. S. boulardii exerts its protective mechanisms by binding and neutralizing enteric pathogens or their toxins, by reducing inflammation and by inducing the secretion of sIgA. Although several S. cerevisiae strains have proven probiotic potential in both humans and animals, only S. boulardii is currently licensed for use in humans. Recently, some researchers started using S. boulardii as heterologous protein expression systems. Combined with their probiotic activity, the use of these strains as prophylactic and therapeutic proteins carriers might result in a positive combined effort to fight specific diseases. Here, we provide an overview of the current use of S. cerevisiae strains as probiotics and their mechanisms of action. We also discuss their potential to produce molecules with biotherapeutic application and the advantages and hurdles of this approach. Finally, we suggest future directions and alternatives for which the combined effort of specific immunomodulatory effects of probiotic S. cerevisiae strains and ability to express desired foreign genes would find a practical application.

  4. Quantification of bioavailable chlortetracycline in pig feces using a bacterial whole-cell biosensor

    DEFF Research Database (Denmark)

    Hansen, L. H.; Aarestrup, Frank Møller; Sørensen, S. J.

    2002-01-01

    Bacterial whole-cell biosensors were used to measure the concentration of chlortetracycline (CTC) in the feces of pigs. In this study, the Escherichia coli biosensor used has a detection limit of 0.03 mg/kg CTC in pig feces. The tetracycline concentration was correlated with the appearance...... in the feces, to within the same order of magnitude as the total coliform count. The high level of tetracycline resistance was maintained in spite of the declining concentration of tetracycline. (C) 2002 Elsevier Science B.V. All rights reserved....

  5. Role of Sulfhydryl Sites on Bacterial Cell Walls in the Biosorption, Mobility and Bioavailability of Mercury and Uranium

    Energy Technology Data Exchange (ETDEWEB)

    Myneni, Satish C. [Princeton Univ., NJ (United States); Mishra, Bhoopesh [Princeton Univ., NJ (United States); Fein, Jeremy [Princeton Univ., NJ (United States)

    2009-04-01

    The goal of this exploratory study is to provide a quantitative and mechanistic understanding of the impact of bacterial sulfhydryl groups on the bacterial uptake, speciation, methylation and bioavailability of Hg and redox changes of uranium. The relative concentration and reactivity of different functional groups present on bacterial surfaces will be determined, enabling quantitative predictions of the role of biosorption of Hg under the physicochemical conditions found at contaminated DOE sites.The hypotheses we propose to test in this investigation are as follows- 1) Sulfhydryl groups on bacterial cell surfaces modify Hg speciation and solubility, and play an important role, specifically in the sub-micromolar concentration ranges of metals in the natural and contaminated systems. 2) Sulfhydryl binding of Hg on bacterial surfaces significantly influences Hg transport into the cell and the methylation rates by the bacteria. 3) Sulfhydryls on cell membranes can interact with hexavalent uranium and convert to insoluble tetravalent species. 4) Bacterial sulfhydryl surface groups are inducible by the presence of metals during cell growth. Our studies focused on the first hypothesis, and we examined the nature of sulfhydryl sites on three representative bacterial species: Bacillus subtilis, a common gram-positive aerobic soil species; Shewanella oneidensis, a facultative gram-negative surface water species; and Geobacter sulfurreducens, an anaerobic iron-reducing gram-negative species that is capable of Hg methylation; and at a range of Hg concentration (and Hg:bacterial concentration ratio) in which these sites become important. A summary of our findings is as follows- Hg adsorbs more extensively to bacteria than other metals. Hg adsorption also varies strongly with pH and chloride concentration, with maximum adsorption occurring under circumneutral pH conditions for both Cl-bearing and Cl-free systems. Under these conditions, all bacterial species tested exhibit

  6. Bacterial Cellulose Shifts Transcriptome and Proteome of Cultured Endothelial Cells Towards Native Differentiation.

    Science.gov (United States)

    Feil, Gerhard; Horres, Ralf; Schulte, Julia; Mack, Andreas F; Petzoldt, Svenja; Arnold, Caroline; Meng, Chen; Jost, Lukas; Boxleitner, Jochen; Kiessling-Wolf, Nicole; Serbest, Ender; Helm, Dominic; Kuster, Bernhard; Hartmann, Isabel; Korff, Thomas; Hahne, Hannes

    2017-09-01

    Preserving the native phenotype of primary cells in vitro is a complex challenge. Recently, hydrogel-based cellular matrices have evolved as alternatives to conventional cell culture techniques. We developed a bacterial cellulose-based aqueous gel-like biomaterial, dubbed Xellulin, which mimics a cellular microenvironment and seems to maintain the native phenotype of cultured and primary cells. When applied to human umbilical vein endothelial cells (HUVEC), it allowed the continuous cultivation of cell monolayers for more than one year without degradation or dedifferentiation. To investigate the impact of Xellulin on the endothelial cell phenotype in detail, we applied quantitative transcriptomics and proteomics and compared the molecular makeup of native HUVEC, HUVEC on collagen-coated Xellulin and collagen-coated cell culture plastic (polystyrene).Statistical analysis of 12,475 transcripts and 7831 proteins unveiled massive quantitative differences of the compared transcriptomes and proteomes. K -means clustering followed by network analysis showed that HUVEC on plastic upregulate transcripts and proteins controlling proliferation, cell cycle and protein biosynthesis. In contrast, HUVEC on Xellulin maintained, by and large, the expression levels of genes supporting their native biological functions and signaling networks such as integrin, receptor tyrosine kinase MAP/ERK and PI3K signaling pathways, while decreasing the expression of proliferation associated proteins. Moreover, CD34-an endothelial cell differentiation marker usually lost early during cell culture - was re-expressed within 2 weeks on Xellulin but not on plastic. And HUVEC on Xellulin showed a significantly stronger functional responsiveness to a prototypic pro-inflammatory stimulus than HUVEC on plastic.Taken together, this is one of the most comprehensive transcriptomic and proteomic studies of native and propagated HUVEC, which underscores the importance of the morphology of the cellular

  7. Cell cycle arrest and biochemical changes accompanying cell death in harmful dinoflagellates following exposure to bacterial algicide IRI-160AA

    Science.gov (United States)

    Pokrzywinski, Kaytee L.; Tilney, Charles L.; Warner, Mark E.; Coyne, Kathryn J.

    2017-03-01

    Bacteria may play a role in regulating harmful algal blooms, but little is known about the biochemical and physiological changes associated with cell death induced by algicidal bacteria. Previous work characterized an algicidal exudate (IRI-160AA) produced by Shewanella sp. IRI-160 that is effective against dinoflagellates, while having little to no effect on other phytoplankton species in laboratory culture experiments. The objective of this study was to evaluate biochemical changes associated with cell death and impacts on the cell cycle in three dinoflagellate species (Prorocentrum minimum, Karlodinium veneficum and Gyrodinium instriatum) after exposure to IRI-160AA. In this study, IRI-160AA induced cell cycle arrest in all dinoflagellates examined. Several indicators for programmed cell death (PCD) that are often observed in phytoplankton in response to a variety of stressors were also evaluated. Cell death was accompanied by significant increases in DNA degradation, intra- and extracellular ROS concentrations and DEVDase (caspase-3 like) protease activity, which have been associated with PCD in other phytoplankton species. Overall, results of this investigation provide strong evidence that treatment with the bacterial algicide, IRI-160AA results in cell cycle arrest and induces biochemical changes consistent with stress-related cell death responses observed in other phytoplankton.

  8. Response Mechanisms of Bacterial Degraders to Environmental Contaminants on the Level of Cell Walls and Cytoplasmic Membrane

    Directory of Open Access Journals (Sweden)

    Slavomíra Murínová

    2014-01-01

    Full Text Available Bacterial strains living in the environment must cope with the toxic compounds originating from humans production. Surface bacterial structures, cell wall and cytoplasmic membrane, surround each bacterial cell and create selective barriers between the cell interior and the outside world. They are a first site of contact between the cell and toxic compounds. Organic pollutants are able to penetrate into cytoplasmic membrane and affect membrane physiological functions. Bacteria had to evolve adaptation mechanisms to counteract the damage originated from toxic contaminants and to prevent their accumulation in cell. This review deals with various adaptation mechanisms of bacterial cell concerning primarily the changes in cytoplasmic membrane and cell wall. Cell adaptation maintains the membrane fluidity status and ratio between bilayer/nonbilayer phospholipids as well as the efflux of toxic compounds, protein repair mechanisms, and degradation of contaminants. Low energy consumption of cell adaptation is required to provide other physiological functions. Bacteria able to survive in toxic environment could help us to clean contaminated areas when they are used in bioremediation technologies.

  9. Response mechanisms of bacterial degraders to environmental contaminants on the level of cell walls and cytoplasmic membrane.

    Science.gov (United States)

    Murínová, Slavomíra; Dercová, Katarína

    2014-01-01

    Bacterial strains living in the environment must cope with the toxic compounds originating from humans production. Surface bacterial structures, cell wall and cytoplasmic membrane, surround each bacterial cell and create selective barriers between the cell interior and the outside world. They are a first site of contact between the cell and toxic compounds. Organic pollutants are able to penetrate into cytoplasmic membrane and affect membrane physiological functions. Bacteria had to evolve adaptation mechanisms to counteract the damage originated from toxic contaminants and to prevent their accumulation in cell. This review deals with various adaptation mechanisms of bacterial cell concerning primarily the changes in cytoplasmic membrane and cell wall. Cell adaptation maintains the membrane fluidity status and ratio between bilayer/nonbilayer phospholipids as well as the efflux of toxic compounds, protein repair mechanisms, and degradation of contaminants. Low energy consumption of cell adaptation is required to provide other physiological functions. Bacteria able to survive in toxic environment could help us to clean contaminated areas when they are used in bioremediation technologies.

  10. Plasma of Argon Increases Cell Attachment and Bacterial Decontamination on Different Implant Surfaces.

    Science.gov (United States)

    Canullo, Luigi; Genova, Tullio; Wang, Hom-Lay; Carossa, Stefano; Mussano, Federico

    This in vitro study tested the effects of argon atmospheric pressure dielectric barrier discharge (APDBD) on different implant surfaces with regard to physical changes, bacterial decontamination, and osteoblast adhesion. Seven hundred twenty disks with three different surface topographies-machined (MAC), titanium plasma-sprayed (TPS), and zirconia-blasted and acid-etched (ZRT)-were tested in this experiment. Bacterial adhesion tests were performed repeatedly on a simplified biofilm of Streptococcus mitis. Bacteria were incubated in the presence of the samples, which were subsequently either left untreated as controls or treated with APDBD for 30, 60, and 120 seconds. Samples were then metalized, prior to the recurring acquisition of images using a scanning electronic microscope (SEM). Protein adsorption, surface wettability, and early biologic response were determined for both treated (120 seconds) and untreated implant surfaces. For depicting the eukaryotic cell behavior, preosteoblastic murine cells were used. Cells were conveniently stained, and nuclei were counted. Cell viability was assessed by a chemiluminescent assay at 1, 2, and 3 days. On all treated samples, values of the contact angle measurements were lower than 10 degrees. The untreated samples showed values of contact angle of 80, 100, and 110 degrees, respectively, for MAC, TPS, and ZRT. The protein adsorption on TPS and ZRT was significantly increased after the plasma of argon treatment. However, no significant effect was noted on the MAC disks. The number and the cell spreading area of adherent osteoblasts significantly increased in all treated surfaces. Nonetheless, argon treatment did not influence the osteoblast proliferation and viability at different time points. Bacteria adhesion was significantly reduced, even after 60 seconds of argon treatment. Preliminary data showed that argon atmospheric pressure dielectric barrier discharge disinfected the implant surface, with potential to promote

  11. Bacterial cell surface properties: role of loosely bound extracellular polymeric substances (LB-EPS).

    Science.gov (United States)

    Zhao, Wenqiang; Yang, Shanshan; Huang, Qiaoyun; Cai, Peng

    2015-04-01

    This study investigated the effect of loosely bound extracellular polymeric substances (LB-EPS) on the comprehensive surface properties of four bacteria (Bacillus subtilis, Streptococcus suis, Escherichia coli and Pseudomonas putida). The removal of LB-EPS from bacterial surfaces by high-speed centrifugation (12,000×g) was confirmed by SEM images. Viability tests showed that the percentages of viable cells ranged from 95.9% to 98.0%, and no significant difference was found after treatment (P>0.05). FTIR spectra revealed the presence of phosphodiester, carboxylic, phosphate, and amino functional groups on bacteria surfaces, and the removal of LB-EPS did not alter the types of cell surface functional groups. Potentiometric titration results suggested the total site concentrations on the intact bacteria were higher than those on LB-EPS free bacteria. Most of the acidity constants (pKa) were almost identical, except the increased pKa values of phosphodiester groups on LB-EPS free S. suis and E. coli surfaces. The electrophoretic mobilities and hydrodynamic diameters of the intact and LB-EPS free bacteria were statistically unchanged (P>0.05), indicating LB-EPS had no influence on the net surface charges and size distribution of bacteria. However, LB-ESP could enhance cell aggregation processes. The four LB-EPS free bacteria all exhibited fewer hydrophobicity values (26.1-65.0%) as compared to the intact cells (47.4-69.3%), suggesting the removal of uncharged nonpolar compounds (e.g., carbohydrates) in LB-EPS. These findings improve our understanding of the changes in cell surface characterizations induced by LB-EPS, and have important implications for assessing the role of LB-EPS in bacterial adhesion and transport behaviors. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Probing interaction of Gram-positive and Gram-negative bacterial cells with ZnO nanorods

    International Nuclear Information System (INIS)

    Jain, Aanchal; Bhargava, Richa; Poddar, Pankaj

    2013-01-01

    In the present work, the physiological effects of the ZnO nanorods on the Gram positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Escherichia coli and Aerobacter aerogenes) bacterial cells have been studied. The analysis of bacterial growth curves for various concentrations of ZnO nanorods indicates that Gram positive and Gram negative bacterial cells show inhibition at concentrations of ∼ 64 and ∼ 256 μg/mL respectively. The marked difference in susceptibility towards nanorods was also validated by spread plate and disk diffusion methods. In addition, the scanning electron micrographs show a clear damage to the cells via changed morphology of the cells from rod to coccoid etc. The confocal optical microscopy images of these cells also demonstrate the reduction in live cell count in the presence of ZnO nanorods. These, results clearly indicate that the antibacterial activity of ZnO nanorods is higher towards Gram positive bacterium than Gram negative bacterium which indicates that the structure of the cell wall might play a major role in the interaction with nanostructured materials and shows high sensitivity to the particle concentration. Highlights: ► Effect of ZnO nanorods on the growth cycles of four bacterial strains. ► A relation has been established between growth rate of bacteria and concentration. ► Serious damage in the morphology of bacterial cells in the presence of ZnO nanorods. ► Microscopic studies to see the time dependent effect on bacterial cells

  13. Accumulation of Poly(3-hydroxybutyrate Helps Bacterial Cells to Survive Freezing.

    Directory of Open Access Journals (Sweden)

    Stanislav Obruca

    Full Text Available Accumulation of polyhydroxybutyrate (PHB seems to be a common metabolic strategy adopted by many bacteria to cope with cold environments. This work aimed at evaluating and understanding the cryoprotective effect of PHB. At first a monomer of PHB, 3-hydroxybutyrate, was identified as a potent cryoprotectant capable of protecting model enzyme (lipase, yeast (Saccharomyces cerevisiae and bacterial cells (Cupriavidus necator against the adverse effects of freezing-thawing cycles. Further, the viability of the frozen-thawed PHB accumulating strain of C. necator was compared to that of the PHB non-accumulating mutant. The presence of PHB granules in cells was revealed to be a significant advantage during freezing. This might be attributed to the higher intracellular level of 3-hydroxybutyrate in PHB accumulating cells (due to the action of parallel PHB synthesis and degradation, the so-called PHB cycle, but the cryoprotective effect of PHB granules seems to be more complex. Since intracellular PHB granules retain highly flexible properties even at extremely low temperatures (observed by cryo-SEM, it can be expected that PHB granules protect cells against injury from extracellular ice. Finally, thermal analysis indicates that PHB-containing cells exhibit a higher rate of transmembrane water transport, which protects cells against the formation of intracellular ice which usually has fatal consequences.

  14. Accumulation of Poly(3-hydroxybutyrate) Helps Bacterial Cells to Survive Freezing

    Science.gov (United States)

    Krzyzanek, Vladislav; Mravec, Filip; Hrubanova, Kamila; Samek, Ota; Kucera, Dan; Benesova, Pavla; Marova, Ivana

    2016-01-01

    Accumulation of polyhydroxybutyrate (PHB) seems to be a common metabolic strategy adopted by many bacteria to cope with cold environments. This work aimed at evaluating and understanding the cryoprotective effect of PHB. At first a monomer of PHB, 3-hydroxybutyrate, was identified as a potent cryoprotectant capable of protecting model enzyme (lipase), yeast (Saccharomyces cerevisiae) and bacterial cells (Cupriavidus necator) against the adverse effects of freezing-thawing cycles. Further, the viability of the frozen–thawed PHB accumulating strain of C. necator was compared to that of the PHB non-accumulating mutant. The presence of PHB granules in cells was revealed to be a significant advantage during freezing. This might be attributed to the higher intracellular level of 3-hydroxybutyrate in PHB accumulating cells (due to the action of parallel PHB synthesis and degradation, the so-called PHB cycle), but the cryoprotective effect of PHB granules seems to be more complex. Since intracellular PHB granules retain highly flexible properties even at extremely low temperatures (observed by cryo-SEM), it can be expected that PHB granules protect cells against injury from extracellular ice. Finally, thermal analysis indicates that PHB-containing cells exhibit a higher rate of transmembrane water transport, which protects cells against the formation of intracellular ice which usually has fatal consequences. PMID:27315285

  15. Scientific and Regulatory Policy Committee Points-to-consider Paper*: Drug-induced Vascular Injury Associated with Nonsmall Molecule Therapeutics in Preclinical Development: Part I. Biotherapeutics.

    Science.gov (United States)

    Frazier, Kendall S; Engelhardt, Jeffery A; Fant, Pierluigi; Guionaud, Silvia; Henry, Scott P; Leach, Michael W; Louden, Calvert; Scicchitano, Marshall S; Weaver, James L; Zabka, Tanja S

    2015-10-01

    Drug-induced vascular injury (DIVI) is a recurrent challenge in the development of novel pharmaceutical agents. Although DIVI in laboratory animal species has been well characterized for vasoactive small molecules, there is little available information regarding DIVI associated with biotherapeutics such as peptides/proteins or antibodies. Because of the uncertainty about whether DIVI in preclinical studies is predictive of effects in humans and the lack of robust biomarkers of DIVI, preclinical DIVI findings can cause considerable delays in or even halt development of promising new drugs. This review discusses standard terminology, characteristics, and mechanisms of DIVI associated with biotherapeutics. Guidance and points to consider for the toxicologist and pathologist facing preclinical cases of biotherapeutic-related DIVI are outlined, and examples of regulatory feedback for each of the mechanistic types of DIVI are included to provide insight into risk assessment. © 2015 by The Author(s).

  16. Spatiotemporal development of the bacterial community in a tubular longitudinal microbial fuel cell

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jung Rae; Premier, Giuliano C. [Glamorgan Univ., Pontypridd (United Kingdom). Faculty of Advnaced Technology; Beecroft, Nelli J.; Avignone-Rossa, Claudio [Surrey Univ., Guildford (United Kingdom). Microbial Sciences; Varcoe, John R.; Slade, Robert C.T. [Surrey Univ., Guildford (United Kingdom). Chemical Sciences; Dinsdale, Richard M.; Guwy, Alan J. [Glamorgan Univ., Pontypridd (United Kingdom). Faculty of Health, Sport and Science; Thumser, Alfred [Surrey Univ., Guildford (United Kingdom). Biochemical Sciences

    2011-05-15

    The spatiotemporal development of a bacterial community in an exoelectrogenic biofilm was investigated in sucrose-fed longitudinal tubular microbial fuel cell reactors, consisting of two serially connected modules. The proportional changes in the microbial community composition were assessed by polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) and DNA sequencing in order to relate them to the performance and stability of the bioelectrochemical system. The reproducibility of duplicated reactors, evaluated by cluster analysis and Jaccard's coefficient, shows 80-90% similarity in species composition. Biofilm development through fed-batch start-up and subsequent stable continuous operation results in a population shift from {gamma}-Proteobacteria- and Bacteroidetes- to Firmicutes-dominated communities, with other diverse species present at much lower relative proportions. DGGE patterns were analysed by range-weighted richness (Rr) and Pareto-Lorenz evenness distribution curves to investigate the evolution of the bacterial community. The first modules shifted from dominance by species closely related to Bacteroides graminisolvens, Raoultella ornithinolytica and Klebsiella sp. BM21 at the start of continuous-mode operation to a community dominated by Paludibacter propionicigenes-, Lactococcus sp.-, Pantoea agglomerans- and Klebsiella oxytoca-related species with stable power generation (6.0 W/m{sup 3}) at day 97. Operational strategies that consider the dynamics of the population will provide useful parameters for evaluating system performance in the practical application of microbial fuel cells. (orig.)

  17. Metabolic Regulation of a Bacterial Cell System with Emphasis on Escherichia coli Metabolism

    Science.gov (United States)

    Shimizu, Kazuyuki

    2013-01-01

    It is quite important to understand the overall metabolic regulation mechanism of bacterial cells such as Escherichia coli from both science (such as biochemistry) and engineering (such as metabolic engineering) points of view. Here, an attempt was made to clarify the overall metabolic regulation mechanism by focusing on the roles of global regulators which detect the culture or growth condition and manipulate a set of metabolic pathways by modulating the related gene expressions. For this, it was considered how the cell responds to a variety of culture environments such as carbon (catabolite regulation), nitrogen, and phosphate limitations, as well as the effects of oxygen level, pH (acid shock), temperature (heat shock), and nutrient starvation. PMID:25937963

  18. Comparison of bacterial cells and amine-functionalized abiotic surfaces as support for Pd nanoparticle synthesis

    DEFF Research Database (Denmark)

    De Corte, Simon; Bechstein, Stefanie; Lokanathan, Arcot R.

    2013-01-01

    An increasing demand for catalytic Pd nanoparticles has motivated the search for sustainable production methods. An innovative approach uses bacterial cells as support material for synthesizing Pd nanoparticles by reduction of Pd(II) with e.g. hydrogen or formate. Nevertheless, drawbacks...... nanoparticles, and that abiotic surfaces could support the Pd particle synthesis as efficiently as bacteria. In this study, we explore the possibility of replacing bacteria with amine-functionalized materials, and we compare different functionalization strategies. Pd nanoparticles formed on the support...... on these surfaces was higher than for Pd particles formed on Shewanella oneidensis cells. Smaller Pd nanoparticles generally have better catalytic properties, and previous studies have shown that the particle size can be lowered by increasing the amount of support material used during Pd particle formation. However...

  19. Simultaneous determination of gene expression and bacterial identity in single cells in defined mixtures of pure cultures

    DEFF Research Database (Denmark)

    Poulsen, Lars K.; Dalton, Helen M.; Angels, Mark

    1997-01-01

    A protocol was developed to achieve the simultaneous determination of gene expression and bacterial identity at the level of single cells: a chromogenic beta-galactosidase activity assay was combined with in situ hybridization of Fluorescently labelled oligonucleotide probes to rRNA. The method...... allows monitoring of gene expression and quantification of beta-galactosidase activity in single cells....

  20. Vancomycin added to the wash solution of the cell-saver. Effect on bacterial contamination.

    Science.gov (United States)

    Perez-Ferrer, A; Gredilla-Díaz, E; de Vicente-Sánchez, J; Navarro-Suay, R; Gilsanz-Rodríguez, F

    2017-04-01

    The aim of this study is to test whether the addition of a low-dose of antibiotic (vancomycin) to the wash solution (saline) of the cell-saver reduces the incidence of bacterial contamination of the autologous red blood cell (RBCs) concentrate recovered. Experimental, randomized, double-blind, parallel group study performed on 20 consecutive patients scheduled for posterior spinal fusion surgery. Intraoperative bleeding was processed through a cell-saver: HaemoLite ® 2+, in which the RBCs were washed according to randomization group, with saline (control group) or saline+10μg/ml -1 vancomycin (vanco group). Data regarding age, weight, processed and recovered volume, blood count, blood culture, and vancomycin concentration in RBCs concentrates obtained and incidence of fever after reinfusion were collected. Processed volume was 843±403ml and recovered volume 121±29ml, with haemoglobin concentration 10.4±5.0g/dl -1 and haematocrit 29.1±15.9% (mean±SD). Recovered RBC concentrate cultures were positive for coagulase-negative Staphylococcus in 5 cases (50%) of the control group while all cultures were negative in the vanco group (P=.016). The difference between the theoretical concentration of vancomycin administered and the concentration determined in the recovered RBC concentrate was 1.31μg/ml -1 (95% CI 1.19 to 1.43; P=.074). The addition of vancomycin at a concentration of 10ug/ml -1 to the wash solution of the cell-saver achieved similar concentrations in the autologous blood concentrate recovered allowing for bacterial removal, with negative blood cultures in all cases. Copyright © 2016 Sociedad Española de Anestesiología, Reanimación y Terapéutica del Dolor. Publicado por Elsevier España, S.L.U. All rights reserved.

  1. Fluorescence-Activated Cell Sorting of Live Versus Dead Bacterial Cells and Spores

    Science.gov (United States)

    Bernardini, James N.; LaDuc, Myron T.; Diamond, Rochelle; Verceles, Josh

    2012-01-01

    This innovation is a coupled fluorescence-activated cell sorting (FACS) and fluorescent staining technology for purifying (removing cells from sampling matrices), separating (based on size, density, morphology, and live versus dead), and concentrating cells (spores, prokaryotic, eukaryotic) from an environmental sample.

  2. Maintenance and assessment of cell viability in formulation of non-sporulating bacterial inoculants.

    Science.gov (United States)

    Berninger, Teresa; González López, Óscar; Bejarano, Ana; Preininger, Claudia; Sessitsch, Angela

    2018-03-01

    The application of beneficial, plant-associated microorganisms is a sustainable approach to improving crop performance in agriculture. However, microbial inoculants are often susceptible to prolonged periods of storage and deleterious environmental factors, which negatively impact their viability and ultimately limit efficacy in the field. This particularly concerns non-sporulating bacteria. To overcome this challenge, the availability of protective formulations is crucial. Numerous parameters influence the viability of microbial cells, with drying procedures generally being among the most critical ones. Thus, technological advances to attenuate the desiccation stress imposed on living cells are key to successful formulation development. In this review, we discuss the core aspects important to consider when aiming at high cell viability of non-sporulating bacteria to be applied as microbial inoculants in agriculture. We elaborate the suitability of commonly applied drying methods (freeze-drying, vacuum-drying, spray-drying, fluidized bed-drying, air-drying) and potential measures to prevent cell damage from desiccation (externally applied protectants, stress pre-conditioning, triggering of exopolysaccharide secretion, 'helper' strains). Furthermore, we point out methods for assessing bacterial viability, such as colony counting, spectrophotometry, microcalorimetry, flow cytometry and viability qPCR. Choosing appropriate technologies for maintenance of cell viability and evaluation thereof will render formulation development more efficient. This in turn will aid in utilizing the vast potential of promising, plant beneficial bacteria as sustainable alternatives to standard agrochemicals. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  3. Bacterial whole-cell biocatalysts by surface display of enzymes: toward industrial application.

    Science.gov (United States)

    Schüürmann, Jan; Quehl, Paul; Festel, Gunter; Jose, Joachim

    2014-10-01

    Despite the first report on the bacterial display of a recombinant peptide appeared almost 30 years ago, industrial application of cells with surface-displayed enzymes is still limited. To display an enzyme on the surface of a living cell bears several advantages. First of all, neither the substrate nor the product of the enzymatic reaction needs to cross a membrane barrier. Second, the enzyme being linked to the cell can be separated from the reaction mixture and hence the product by simple centrifugation. Transfer to a new substrate preparation results in multiple cycles of enzymatic conversion. Finally, the anchoring in a matrix, in this case, the cell envelope stabilizes the enzyme and makes it less accessible to proteolytic degradation and material adsorption resulting in continuous higher activities. These advantages in common need to balance some disadvantages before this application can be taken into account for industrial processes, e.g., the exclusion of the enzyme from the cellular metabolome and hence from redox factors or other co-factors that need to be supplied. Therefore, this digest describes the different systems in Gram-positive and Gram-negative bacteria that have been used for the surface display of enzymes so far and focuses on examples among these which are suitable for industrial purposes or for the production of valuable resources, not least in order to encourage a broader application of whole-cell biocatalysts with surface-displayed enzymes.

  4. Synthesis and in vitro safety assessment of magnetic bacterial cellulose with porcine aortic smooth muscle cells.

    Science.gov (United States)

    Pastrana, Homero F; Cooper, Christy L; Alucozai, Milad; Reece, Lisa M; Avila, Alba G; Allain, Jean Paul

    2016-11-01

    Bacterial cellulose (BC) has been used as a scaffold for tissue regeneration (TR). Improving functional TR requires highly selective strategies for specific cell attraction. Embedding iron oxide nanoparticles into a BC matrix can drive magnetically labeled cells to specific tissues where they may begin to heal injured tissue. This article focuses on characterization and in vitro toxicity assessment of magnetic BC (MBC). We proposed to detect the production of radical oxygen species (ROS), esterase activity, and apoptosis to study cytotoxic interactions of MBC within its bioenvironment. Morphological characterization was performed using scanning electron microscopy where evidence shows that the diameter of MBC fibers compared to BC fibers was 33% smaller, and the pore areas were 25% bigger. Cytotoxicity assays in porcine aortic smooth muscle cells exposed for 24 hours to BC, MBC, and poly(ethylene glycol)-coated MBC (MBC-PEG) reveals 96% viability and 9% ROS production for MBC-PEG. In contrast, 25% of cells exposed to MBC were apoptotic, suggesting that even when the cells were metabolically active, MBC can induce damage. These outcomes support the need for more integral assessment in the hopes of assessing the potential biosafety and uses of nanocomposites for TR. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2801-2809, 2016. © 2016 Wiley Periodicals, Inc.

  5. Cytolethal distending toxin: a conserved bacterial genotoxin that blocks cell cycle progression, leading to apoptosis of a broad range of mammalian cell lineages.

    Science.gov (United States)

    Jinadasa, Rasika N; Bloom, Stephen E; Weiss, Robert S; Duhamel, Gerald E

    2011-07-01

    Cytolethal distending toxin (CDT) is a heterotrimeric AB-type genotoxin produced by several clinically important Gram-negative mucocutaneous bacterial pathogens. Irrespective of the bacterial species of origin, CDT causes characteristic and irreversible cell cycle arrest and apoptosis in a broad range of cultured mammalian cell lineages. The active subunit CdtB has structural homology with the phosphodiesterase family of enzymes including mammalian DNase I, and alone is necessary and sufficient to account for cellular toxicity. Indeed, mammalian cells treated with CDT initiate a DNA damage response similar to that elicited by ionizing radiation-induced DNA double strand breaks resulting in cell cycle arrest and apoptosis. The mechanism of CDT-induced apoptosis remains incompletely understood, but appears to involve both p53-dependent and -independent pathways. While epithelial, endothelial and fibroblast cell lines respond to CDT by undergoing arrest of cell cycle progression resulting in nuclear and cytoplasmic distension that precedes apoptotic cell death, cells of haematopoietic origin display rapid apoptosis following a brief period of cell cycle arrest. In this review, the ecology of pathogens producing CDT, the molecular biology of bacterial CDT and the molecular mechanisms of CDT-induced cytotoxicity are critically appraised. Understanding the contribution of a broadly conserved bacterial genotoxin that blocks progression of the mammalian cell cycle, ultimately causing cell death, should assist with elucidating disease mechanisms for these important pathogens.

  6. Reactions of human dental pulp cells to capping agents in the presence or absence of bacterial exposure.

    Science.gov (United States)

    Cai, Shiwei; Zhang, Wenjian; Tribble, Gena; Chen, Wei

    2017-01-01

    An ideal pulp-capping agent needs to have good biocompatibility and promote reparative dentinogenesis. Although the effects of capping agents on healthy pulp are known, limited data regarding their effects on bacterial contaminated pulp are available. This study aimed to evaluate the reaction of contaminated pulps to various capping agents to assist clinicians in making informed decisions. Human dental pulp (HDP) cell cultures were developed from extracted human molars. The cells were exposed to a bacterial cocktail comprising Porphyromonas gingivalis, Prevotella intermedia, and Streptococcus gordonii before being cocultured with capping agents such as mineral trioxide aggregate (MTA) Portland cement (PC), and Dycal. HDP cell proliferation was assayed by MTS colorimetric cell proliferation assay, and its differentiation was evaluated by real-time PCR for detecting alkaline phosphatase, dentin sialophosphoprotein, and osteocalcin expressions. MTA and PC had no apparent effect, whereas Dycal inhibited HDP cell proliferation. PC stimulated HDP cell differentiation, particularly when they were exposed to bacteria. MTA and Dycal inhibited differentiation, regardless of bacterial infection. In conclusion, PC was the most favorable agent, followed by MTA, and Dycal was the least favorable agent for supporting the functions of bacterial compromised pulp cells.

  7. Quorum sensing integrates environmental cues, cell density and cell history to control bacterial competence.

    Science.gov (United States)

    Moreno-Gámez, Stefany; Sorg, Robin A; Domenech, Arnau; Kjos, Morten; Weissing, Franz J; van Doorn, G Sander; Veening, Jan-Willem

    2017-10-11

    Streptococcus pneumoniae becomes competent for genetic transformation when exposed to an autoinducer peptide known as competence-stimulating peptide (CSP). This peptide was originally described as a quorum-sensing signal, enabling individual cells to regulate competence in response to population density. However, recent studies suggest that CSP may instead serve as a probe for sensing environmental cues, such as antibiotic stress or environmental diffusion. Here, we show that competence induction can be simultaneously influenced by cell density, external pH, antibiotic-induced stress, and cell history. Our experimental data is explained by a mathematical model where the environment and cell history modify the rate at which cells produce or sense CSP. Taken together, model and experiments indicate that autoinducer concentration can function as an indicator of cell density across environmental conditions, while also incorporating information on environmental factors or cell history, allowing cells to integrate cues such as antibiotic stress into their quorum-sensing response. This unifying perspective may apply to other debated quorum-sensing systems.Peptide CSP regulates natural competence in pneumococci and has been proposed as a quorum-sensing signal or a probe for sensing environmental cues. Here, the authors show that CSP levels can indeed act as an indicator of cell density and also incorporate information on environmental factors or cell history.

  8. Micro Corona Ionizer as an Ozone Source for Bacterial Cell Lysis

    Science.gov (United States)

    Lee, Eun-Hee; Lim, Hyun Jeong; Chua, Beelee; Son, Ahjeong

    2015-04-01

    DNA extraction is a critical process of DNA assays including polymerase chain reaction (PCR), microarrays, molecular cloning, and DNA hybridization which has been well established and can be implemented by commercial kits. DNA extraction involves cell lysis, precipitation, and purification through the combination of physical and chemical processes. Cell lysis is essential to high DNA recovery yield which can be achieved via a variety of physical, chemical, and enzymatic methods. However, these methods were originally developed for bioassays that were labor intensive, time consuming, and vulnerable to contamination and inhibition. Here, we proposed to employ a micro corona ionizer as an ozone source to lyse bacterial cells. Ozone has been well known and used as a disinfectant which allows cell lysis and DNA extraction. Previously, we have shown that a micro corona ionizer is capable of generating a significant amount of ozone. In this study, we employed the micro corona ionizer for the bacterial cell lysis which consists of a 50 μm diameter cantilever wire as the discharge cathode and a 50 μm thick copper foil as anode. Applied voltages varied from 1900 to 2200 V with corresponding corona currents from 16 to 28 μA. The resultant ozone (concentration > 0.14 ppm) generated from the micro corona ionizer was bubbled into the sample via a miniature pump. We demonstrated the cell lysis of Pseudomonas putida as the target bacterium using the micro corona ionizer. At a flow rate of 38 ml/min and applied corona voltage of 2000 V, 98.5 ± 0.2% lysis (normalized to sonication result) was achieved after 10 min. In comparison, untreated and air-treated samples showed normalized % lysis of 11.9 ± 2.4 and 36.1 ± 1.7%, respectively. We also showed that the cell lysis efficiency could be significantly increased by increasing the flow rate and the applied corona voltage. By comparing the experimental results for continuous and pulsed treatment, we verified that the percentage of

  9. Cell proliferation, viability, and in vitro differentiation of equine mesenchymal stem cells seeded on bacterial cellulose hydrogel scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Favi, Pelagie M.; Benson, Roberto S. [Department of Materials Science and Engineering, College of Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Neilsen, Nancy R. [Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Hammonds, Ryan L. [Department of Materials Science and Engineering, College of Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Bates, Cassandra C. [Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Stephens, Christopher P. [Department of Surgery, Graduate School of Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Center for Materials Processing, University of Tennessee, Knoxville, TN 37996 (United States); Dhar, Madhu S., E-mail: mdhar@utk.edu [Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States)

    2013-05-01

    The culture of multipotent mesenchymal stem cells on natural biopolymers holds great promise for treatments of connective tissue disorders such as osteoarthritis. The safety and performance of such therapies relies on the systematic in vitro evaluation of the developed stem cell-biomaterial constructs prior to in vivo implantation. This study evaluates bacterial cellulose (BC), a biocompatible natural polymer, as a scaffold for equine-derived bone marrow mesenchymal stem cells (EqMSCs) for application in bone and cartilage tissue engineering. An equine model was chosen due to similarities in size, load and types of joint injuries suffered by horses and humans. Lyophilized and critical point dried BC hydrogel scaffolds were characterized using scanning electron microscopy (SEM) to confirm nanostructure morphology which demonstrated that critical point drying induces fibre bundling unlike lyophilisation. EqMSCs positively expressed the undifferentiated pluripotent mesenchymal stem cell surface markers CD44 and CD90. The BC scaffolds were shown to be cytocompatible, supporting cellular adhesion and proliferation, and allowed for osteogenic and chondrogenic differentiation of EqMSCs. The cells seeded on the BC hydrogel were shown to be viable and metabolically active. These findings demonstrate that the combination of a BC hydrogel and EqMSCs are promising constructs for musculoskeletal tissue engineering applications. - Highlights: ► Critical point drying induces fibre bundling unlike lyophilisation. ► Cells positively expressed undifferentiated pluripotent stem cell markers. ► BCs were cytocompatible, supported cell adhesion, proliferation and differentiation ► Cells seeded on BC scaffolds were viable and metabolically active. ► Findings demonstrate that BC and EqMSCs are promising tissue engineered constructs.

  10. Bacterial and mammalian cells adhesion to tantalum-decorated micro-/nano-structured titanium.

    Science.gov (United States)

    Zhu, Yu; Gu, Yingxin; Qiao, Shichong; Zhou, Linyi; Shi, Junyu; Lai, Hongchang

    2017-03-01

    Microorganisms are frequently introduced to dental implants during surgery and start the race for the surface with host cells before osseointegration occurs. The aim of the study was to endow implant surfaces with biological functions that reliably select cells over microbes. Nano-structured tantalum (Ta) has exhibited excellent compatibility. Thus, nano-structured Ta films were deposited on the sand-blasted, large grit, and acid-etched (SLA) titanium by the magnetron sputtering method, thus forming hierarchical micro-/nano-structured surfaces. No obvious Ta release confirmed the robustness of the deposited layer probably arising from the stable Ta 2 O 5 . Moreover, Ta-modified surfaces not only improved the initial adhesion and spreading of rat bone mesenchymal stem cells (rBMSCs), but also exhibited good antibacterial activities towards Streptococcus mutans and Porphyromonas gingivalis. The satisfactory cell-surface interactions on Ta-modified surfaces depended largely on the up-regulation of adhesion-related genes and activation of focal adhesion kinase (FAK), as confirmed by real-time PCR and Western blot. Here, the coculture model was also forwarded to mimic the perioperative bacterial contamination. We found that the adherent cell number and the cell-surface coverage were hampered by bacteria presence on both surfaces. Yet, rBMSCs still attached and spread more readily on Ta-modified surfaces than on SLA titanium surfaces even in coculture with adhering oral pathogens. Our results revealed that Ta-modified micro-/nano-structured surfaces would selectively promote cell-surface rather than bacteria-surface interactions, boding well for the applications for dental implants in possibly infected environments. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 871-878, 2017. © 2016 Wiley Periodicals, Inc.

  11. Seasonal and spatial distribution of bacterial biomass and the percentage of viable cells in a reservoir of Alabama

    Science.gov (United States)

    Tietjen, T.E.; Wetzel, R.G.

    2003-01-01

    Spatial community dynamics of bacterioplankton were evaluated along the length of the former stream channel of Elledge Lake, a small reservoir in western Alabama. The reservoir was strongly stratified from April to October with up to a 10??C temperature difference across the 1 m deep metalimnion. Bacterial biomass was highest during late summer, with a general pattern of increasing abundance from the inflowing river (???10 ??g C l-1) to the dam (???20-30 ??g C l-1). Bacterial numbers also increased following a >10-fold increase in turbidity associated with a major precipitation event, although only ???10% of these cells were viable. The percentage of viable cells generally increased through the stratified period with 50-70% viable cells in late summer. Overall, an average of 38% of bacterial cells were viable, with a range from <20 to 70%. Although these values were similar to those found by others, additional patterns were identified that have not been previously observed: a marked decline in viable cells was found following turbid storm inflows and increases in the percentage of viable cells occurred during spring warming and following autumnal mixing events. Although a modest increase in abundance occurred along the gradient from inflow down-reservoir to the dam, bacterial abundance did not increase near the dam in a pattern coincident with the commonly observed increased algal biomass in the lacustrine portion of reservoir ecosystems. The increases observed in bacterial viability moving from the inflowing rivers towards the dam and later in stratified periods stress the importance of differences in environmental conditions in time and space in regulating bacterial biomass and development, as well as of shifts that would be anticipated accompanying altered hydrological regimes under climatic change.

  12. Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux in a mammalian cell line.

    Directory of Open Access Journals (Sweden)

    Dan M Close

    Full Text Available The bacterial luciferase (lux gene cassette consists of five genes (luxCDABE whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo.Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH(2 was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp from Vibrio harveyi. FMNH(2 supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background.The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.

  13. SLiCE: a novel bacterial cell extract-based DNA cloning method

    Science.gov (United States)

    Zhang, Yongwei; Werling, Uwe; Edelmann, Winfried

    2012-01-01

    We describe a novel cloning method termed SLiCE (Seamless Ligation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from Bacteria Artificial Chromosomes (BACs) or other sources. SLiCE is highly cost effective as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. In addition, the cloning efficiencies and capabilities of these strains can be greatly improved by simple genetic modifications. As an example, we modified the DH10B Escherichia coli strain to express an optimized λ prophage Red recombination system. This strain, termed PPY, facilitates SLiCE with very high efficiencies and demonstrates the versatility of the method. PMID:22241772

  14. SLiCE: a novel bacterial cell extract-based DNA cloning method.

    Science.gov (United States)

    Zhang, Yongwei; Werling, Uwe; Edelmann, Winfried

    2012-04-01

    We describe a novel cloning method termed SLiCE (Seamless Ligation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from Bacteria Artificial Chromosomes (BACs) or other sources. SLiCE is highly cost effective as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. In addition, the cloning efficiencies and capabilities of these strains can be greatly improved by simple genetic modifications. As an example, we modified the DH10B Escherichia coli strain to express an optimized λ prophage Red recombination system. This strain, termed PPY, facilitates SLiCE with very high efficiencies and demonstrates the versatility of the method.

  15. Presence of viral and bacterial organisms in milk and their association with somatic cell counts.

    Science.gov (United States)

    Herlekar, D A; Shashikant, C S; Gurjar, A A; Jayarao, B M

    2013-10-01

    About 20 to 35% of milk samples from cows with intramammary infection or high somatic cell count (SCC) are negative on bacteriological culture analysis. However, little is known about SCC in milk of cows infected with viruses. In the first part of our study, we developed a real-time PCR assay for detection of bovine herpesvirus (BHV) 1, BHV2, and BHV4, and bovine viral diarrhea virus (BVDV) in composite quarter milk samples. A total of 1,479 lactating cows of 1,964 cows in the dairy herd were initially selected because these cows had complete SCC data for at least 3 consecutive test results, of which 139 lactating cows from different lactation age groups were selected randomly and studied extensively. Composite quarter milk samples were collected on 3 alternate days and examined for viruses, SCC, and bacteriological analysis. In total, 10, 28, and 0.7% of the composite quarter milk samples from cows were positive for BHV1, BHV2, and BHV4, respectively; BVDV was not detected in composite quarter milk samples. Bovine herpesvirus was not associated with a particular bacterial species. Our study results indicate that cows positive for BHV in composite quarter milk samples alone are less likely to have elevated SCC compared with cows with bacterial intramammary infection; BHV1, BHV2, and BHV4 are probably not major udder pathogens. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  16. The Effect of Cell Immobilization by Calcium Alginate on Bacterially Induced Calcium Carbonate Precipitation

    Directory of Open Access Journals (Sweden)

    Mostafa Seifan

    2017-10-01

    Full Text Available Microbially induced mineral precipitation is recognized as a widespread phenomenon in nature. A diverse range of minerals including carbonate, sulphides, silicates, and phosphates can be produced through biomineralization. Calcium carbonate (CaCO3 is one of the most common substances used in various industries and is mostly extracted by mining. In recent years, production of CaCO3 by bacteria has drawn much attention because it is an environmentally- and health-friendly pathway. Although CaCO3 can be produced by some genera of bacteria through autotrophic and heterotrophic pathways, the possibility of producing CaCO3 in different environmental conditions has remained a challenge to determine. In this study, calcium alginate was proposed as a protective carrier to increase the bacterial tolerance to extreme environmental conditions. The model showed that the highest concentration of CaCO3 is achieved when the bacterial cells are immobilized in the calcium alginate beads fabricated using 1.38% w/v Na-alginate and 0.13 M CaCl2.

  17. Antibiotic Discovery: Combatting Bacterial Resistance in Cells and in Biofilm Communities

    Directory of Open Access Journals (Sweden)

    Anahit Penesyan

    2015-03-01

    Full Text Available Bacterial resistance is a rapidly escalating threat to public health as our arsenal of effective antibiotics dwindles. Therefore, there is an urgent need for new antibiotics. Drug discovery has historically focused on bacteria growing in planktonic cultures. Many antibiotics were originally developed to target individual bacterial cells, being assessed in vitro against microorganisms in a planktonic mode of life. However, towards the end of the 20th century it became clear that many bacteria live as complex communities called biofilms in their natural habitat, and this includes habitats within a human host. The biofilm mode of life provides advantages to microorganisms, such as enhanced resistance towards environmental stresses, including antibiotic challenge. The community level resistance provided by biofilms is distinct from resistance mechanisms that operate at a cellular level, and cannot be overlooked in the development of novel strategies to combat infectious diseases. The review compares mechanisms of antibiotic resistance at cellular and community levels in the light of past and present antibiotic discovery efforts. Future perspectives on novel strategies for treatment of biofilm-related infectious diseases are explored.

  18. Identification of Bacterial Surface Antigens by Screening Peptide Phage Libraries Using Whole Bacteria Cell-Purified Antisera

    Science.gov (United States)

    Hu, Yun-Fei; Zhao, Dun; Yu, Xing-Long; Hu, Yu-Li; Li, Run-Cheng; Ge, Meng; Xu, Tian-Qi; Liu, Xiao-Bo; Liao, Hua-Yuan

    2017-01-01

    Bacterial surface proteins can be good vaccine candidates. In the present study, we used polyclonal antibodies purified with intact Erysipelothrix rhusiopthiae to screen phage-displayed random dodecapeptide and loop-constrained heptapeptide libraries, which led to the identification of mimotopes. Homology search of the mimotope sequences against E. rhusiopthiae-encoded ORF sequences revealed 14 new antigens that may localize on the surface of E. rhusiopthiae. When these putative surface proteins were used to immunize mice, 9/11 antigens induced protective immunity. Thus, we have demonstrated that a combination of using the whole bacterial cells to purify antibodies and using the phage-displayed peptide libraries to determine the antigen specificities of the antibodies can lead to the discovery of novel bacterial surface antigens. This can be a general approach for identifying surface antigens for other bacterial species. PMID:28184219

  19. Single-cell level based approach to investigate bacterial metabolism during batch industrial fermentation

    DEFF Research Database (Denmark)

    Nierychlo, Marta; Larsen, Poul; Eriksen, Niels T.

    , and performance of Escherichia coli. An insight into glucose and acetate fate on the level of individual cell can provide the type of information which are valuable for the understanding of bacterial metabolism in fermentation process and can shed more light on the differentiation of isogenic fermenting...... can exhibit different phenotypes under specific environmental conditions that show significant differences in physiological parameters from the population average. However, studies concerning segregation of populations into metabolically diversified subpopulations are scarce. Acetate is a product...... of Escherichia coli overflow metabolism when the bacteria are grown under aerobic conditions and glucose is present in excessive concentrations. Acetate accumulation is of the utmost importance in batch fermentation processes as it is an undesirable byproduct that negatively affects growth, physiology...

  20. Coronatine inhibits stomatal closure and delays hypersensitive response cell death induced by nonhost bacterial pathogens

    Directory of Open Access Journals (Sweden)

    Seonghee Lee

    2013-02-01

    Full Text Available Pseudomonas syringae is the most widespread bacterial pathogen in plants. Several strains of P. syringae produce a phytotoxin, coronatine (COR, which acts as a jasmonic acid mimic and inhibits plant defense responses and contributes to disease symptom development. In this study, we found that COR inhibits early defense responses during nonhost disease resistance. Stomatal closure induced by a nonhost pathogen, P. syringae pv. tabaci, was disrupted by COR in tomato epidermal peels. In addition, nonhost HR cell death triggered by P. syringae pv. tabaci on tomato was remarkably delayed when COR was supplemented along with P. syringae pv. tabaci inoculation. Using isochorismate synthase (ICS-silenced tomato plants and transcript profiles of genes in SA- and JA-related defense pathways, we show that COR suppresses SA-mediated defense during nonhost resistance.

  1. Synthesis and Evaluation of Quinazolines as Inhibitors of the Bacterial Cell Division Protein FtsZ.

    Science.gov (United States)

    Nepomuceno, Gabriella M; Chan, Katie M; Huynh, Valerie; Martin, Kevin S; Moore, Jared T; O'Brien, Terrence E; Pollo, Luiz A E; Sarabia, Francisco J; Tadeus, Clarissa; Yao, Zi; Anderson, David E; Ames, James B; Shaw, Jared T

    2015-03-12

    The bacterial cell division protein FtsZ is one of many potential targets for the development of novel antibiotics. Recently, zantrin Z3 was shown to be a cross-species inhibitor of FtsZ; however, its specific interactions with the protein are still unknown. Herein we report the synthesis of analogues that contain a more tractable core structure and an analogue with single-digit micromolar inhibition of FtsZ's GTPase activity, which represents the most potent inhibitor of Escherichia coli FtsZ reported to date. In addition, the zantrin Z3 core has been converted to two potential photo-cross-linking reagents for proteomic studies that could shed light on the molecular interactions between FtsZ and molecules related to zantrin Z3.

  2. Decolorization of industrial synthetic dyes using engineered Pseudomonas putida cells with surface-immobilized bacterial laccase

    Science.gov (United States)

    2012-01-01

    Background Microbial laccases are highly useful in textile effluent dye biodegradation. However, the bioavailability of cellularly expressed or purified laccases in continuous operations is usually limited by mass transfer impediment or enzyme regeneration difficulty. Therefore, this study develops a regenerable bacterial surface-displaying system for industrial synthetic dye decolorization, and evaluates its effects on independent and continuous operations. Results A bacterial laccase (WlacD) was engineered onto the cell surface of the solvent-tolerant bacterium Pseudomonas putida to construct a whole-cell biocatalyst. Ice nucleation protein (InaQ) anchor was employed, and the ability of 1 to 3 tandemly aligned N-terminal repeats to direct WlacD display were compared. Immobilized WlacD was determined to be surface-displayed in functional form using Western blot analysis, immunofluorescence microscopy, flow cytometry, and whole-cell enzymatic activity assay. Engineered P. putida cells were then applied to decolorize the anthraquinone dye Acid Green (AG) 25 and diazo-dye Acid Red (AR) 18. The results showed that decolorization of both dyes is Cu2+- and mediator-independent, with an optimum temperature of 35°C and pH of 3.0, and can be stably performed across a temperature range of 15°C to 45°C. A high activity toward AG25 (1 g/l) with relative decolorization values of 91.2% (3 h) and 97.1% (18 h), as well as high activity to AR18 (1 g/l) by 80.5% (3 h) and 89.0% (18 h), was recorded. The engineered system exhibited a comparably high activity compared with those of separate dyes in a continuous three-round shake-flask decolorization of AG25/AR18 mixed dye (each 1 g/l). No significant decline in decolorization efficacy was noted during first two-rounds but reaction equilibriums were elongated, and the residual laccase activity eventually decreased to low levels. However, the decolorizing capacity of the system was easily retrieved via a subsequent 4-h

  3. Decolorization of industrial synthetic dyes using engineered Pseudomonas putida cells with surface-immobilized bacterial laccase

    Directory of Open Access Journals (Sweden)

    Wang Wei

    2012-06-01

    Full Text Available Abstract Background Microbial laccases are highly useful in textile effluent dye biodegradation. However, the bioavailability of cellularly expressed or purified laccases in continuous operations is usually limited by mass transfer impediment or enzyme regeneration difficulty. Therefore, this study develops a regenerable bacterial surface-displaying system for industrial synthetic dye decolorization, and evaluates its effects on independent and continuous operations. Results A bacterial laccase (WlacD was engineered onto the cell surface of the solvent-tolerant bacterium Pseudomonas putida to construct a whole-cell biocatalyst. Ice nucleation protein (InaQ anchor was employed, and the ability of 1 to 3 tandemly aligned N-terminal repeats to direct WlacD display were compared. Immobilized WlacD was determined to be surface-displayed in functional form using Western blot analysis, immunofluorescence microscopy, flow cytometry, and whole-cell enzymatic activity assay. Engineered P. putida cells were then applied to decolorize the anthraquinone dye Acid Green (AG 25 and diazo-dye Acid Red (AR 18. The results showed that decolorization of both dyes is Cu2+- and mediator-independent, with an optimum temperature of 35°C and pH of 3.0, and can be stably performed across a temperature range of 15°C to 45°C. A high activity toward AG25 (1 g/l with relative decolorization values of 91.2% (3 h and 97.1% (18 h, as well as high activity to AR18 (1 g/l by 80.5% (3 h and 89.0% (18 h, was recorded. The engineered system exhibited a comparably high activity compared with those of separate dyes in a continuous three-round shake-flask decolorization of AG25/AR18 mixed dye (each 1 g/l. No significant decline in decolorization efficacy was noted during first two-rounds but reaction equilibriums were elongated, and the residual laccase activity eventually decreased to low levels. However, the decolorizing capacity of the system was easily retrieved

  4. Bacterial nanocellulose/Nafion composite membranes for low temperature polymer electrolyte fuel cells

    Science.gov (United States)

    Jiang, Gao-peng; Zhang, Jing; Qiao, Jin-li; Jiang, Yong-ming; Zarrin, Hadis; Chen, Zhongwei; Hong, Feng

    2015-01-01

    Novel nanocomposite membranes aimed for both proton-exchange membrane fuel cell (PEMFC) and direct methanol fuel cell (DMFC) are presented in this work. The membranes are based on blending bacterial nanocellulose pulp and Nafion (abbreviated as BxNy, where x and y indicates the mass ratio of bacterial cellulose to Nafion). The structure and properties of BxNy membranes are characterized by FTIR, SEM, TG, DMA and EIS, along with water uptake, swelling behavior and methanol permeability tests. It is found that the BxNy composite membranes with reinforced concrete-like structure show excellent mechanical and thermal stability regardless of annealing. The water uptake plus area and volume swelling ratios are all decreased compared to Nafion membranes. The proton conductivities of pristine and annealed B1N9 are 0.071 and 0.056 S cm-1, respectively, at 30 °C and 100% humidity. Specifically, annealed B1N1 exhibited the lowest methanol permeability of 7.21 × 10-7 cm2 s-1. Through the selectivity analysis, pristine and annealed B1N7 are selected to assemble the MEAs. The performances of annealed B1N7 in PEMFC and DMFC show the maximum power densities of 106 and 3.2 mW cm-2, respectively, which are much higher than those of pristine B1N7 at 25 °C. The performances of the pristine and annealed B1N7 reach a level as high as 21.1 and 20.4 mW cm-2 at 80 °C in DMFC, respectively.

  5. Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion

    DEFF Research Database (Denmark)

    van Wijk, Xander M.; Döhrmann, Simon; Hallstrom, Bjorn

    2017-01-01

    To understand the role of glycosaminoglycans in bacterial cellular invasion, xylosyltransferase-deficient mutants of Chinese hamster ovary (CHO) cells were created using clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-cas9) gene targeting. When...... these mutants were compared to the pgsA745 cell line, a CHO xylosyltransferase mutant generated previously using chemical mutagenesis, an unexpected result was obtained. Bacterial invasion of pgsA745 cells by group B Streptococcus (GBS), group A Streptococcus, and Staphylococcus aureus was markedly reduced...... compared to the invasion of wild-type cells, but newly generated CRISPR-cas9 mutants were only resistant to GBS. Invasion of pgsA745 cells was not restored by transfection with xylosyltransferase, suggesting that an additional mutation conferring panresistance to multiple bacteria was present in pgsA745...

  6. Process development in the QbD paradigm: Role of process integration in process optimization for production of biotherapeutics.

    Science.gov (United States)

    Rathore, Anurag S; Pathak, Mili; Godara, Avinash

    2016-03-01

    Biotherapeutics have become the focus of the pharmaceutical industry due to their proven effectiveness in managing complex diseases. Downstream processes of these molecules consist of several orthogonal, high resolution unit operations designed so as to be able to separate variants having very similar physicochemical properties. Typical process development involves optimization of the individual unit operations based on Quality by Design principles in order to define the design space within which the process can deliver product that meets the predefined specifications. However, limited efforts are dedicated to understanding the interactions between the unit operations. This paper aims to showcase the importance of understanding these interactions and thereby arrive at operating conditions that are optimal for the overall process. It is demonstrated that these are not necessarily same as those obtained from optimization of the individual unit operations. Purification of Granulocyte Colony Stimulating Factor (G-CSF), a biotherapeutic expressed in E. coli., has been used as a case study. It is evident that the suggested approach results in not only higher yield (91.5 vs. 86.4) but also improved product quality (% RP-HPLC purity of 98.3 vs. 97.5) and process robustness. We think that this paper is very relevant to the present times when the biotech industry is in the midst of implementing Quality by Design towards process development. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:355-362, 2016. © 2015 American Institute of Chemical Engineers.

  7. Desialylation of Spermatozoa and Epithelial Cell Glycocalyx Is a Consequence of Bacterial Infection of the Epididymis*

    Science.gov (United States)

    Khosravi, Farhad; Michel, Vera; Galuska, Christina E.; Bhushan, Sudhanshu; Christian, Philipp; Schuppe, Hans-Christian; Pilatz, Adrian; Galuska, Sebastian P.; Meinhardt, Andreas

    2016-01-01

    Urinary tract infections caused by uropathogenic Escherichia coli (UPEC) pathovars belong to the most frequent infections in humans. In men, pathogens can also spread to the genital tract via the continuous ductal system, eliciting bacterial prostatitis and/or epididymo-orchitis. Antibiotic treatment usually clears pathogens in acute epididymitis; however, the fertility of patients can be permanently impaired. Because a premature acrosome reaction was observed in an UPEC epididymitis mouse model, and sialidases on the sperm surface are considered to be activated via proteases of the acrosome, we aimed to investigate whether alterations of the sialome of epididymal spermatozoa and surrounding epithelial cells occur during UPEC infection. In UPEC-elicited acute epididymitis in mice, a substantial loss of N-acetylneuraminic acid residues was detected in epididymal spermatozoa and epithelial cells using combined laser microdissection/HPLC-ESI-MS analysis. In support, a substantial reduction of sialic acid residues bound to the surface of spermatozoa was documented in men with a recent history of E. coli-associated epididymitis. In vitro, such an UPEC induced N-acetylneuraminic acid release from human spermatozoa was effectively counteracted by a sialidase inhibitor. These findings strongly suggest a substantial remodeling of the glycocalyx of spermatozoa and epididymal epithelial cells by endogenous sialidases after a premature acrosome reaction during acute epididymitis. PMID:27339898

  8. Desialylation of Spermatozoa and Epithelial Cell Glycocalyx Is a Consequence of Bacterial Infection of the Epididymis.

    Science.gov (United States)

    Khosravi, Farhad; Michel, Vera; Galuska, Christina E; Bhushan, Sudhanshu; Christian, Philipp; Schuppe, Hans-Christian; Pilatz, Adrian; Galuska, Sebastian P; Meinhardt, Andreas

    2016-08-19

    Urinary tract infections caused by uropathogenic Escherichia coli (UPEC) pathovars belong to the most frequent infections in humans. In men, pathogens can also spread to the genital tract via the continuous ductal system, eliciting bacterial prostatitis and/or epididymo-orchitis. Antibiotic treatment usually clears pathogens in acute epididymitis; however, the fertility of patients can be permanently impaired. Because a premature acrosome reaction was observed in an UPEC epididymitis mouse model, and sialidases on the sperm surface are considered to be activated via proteases of the acrosome, we aimed to investigate whether alterations of the sialome of epididymal spermatozoa and surrounding epithelial cells occur during UPEC infection. In UPEC-elicited acute epididymitis in mice, a substantial loss of N-acetylneuraminic acid residues was detected in epididymal spermatozoa and epithelial cells using combined laser microdissection/HPLC-ESI-MS analysis. In support, a substantial reduction of sialic acid residues bound to the surface of spermatozoa was documented in men with a recent history of E. coli-associated epididymitis. In vitro, such an UPEC induced N-acetylneuraminic acid release from human spermatozoa was effectively counteracted by a sialidase inhibitor. These findings strongly suggest a substantial remodeling of the glycocalyx of spermatozoa and epididymal epithelial cells by endogenous sialidases after a premature acrosome reaction during acute epididymitis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Disinfectants - bacterial cells interactions in the view of hygiene and public health

    Directory of Open Access Journals (Sweden)

    Marta Książczyk

    2015-09-01

    Full Text Available In recent years, the use of biocides has increased rapidly. One common example is triclosan, with wide application in households as well as medical and industrial fields, especially food industry and animal husbandry. Chemical disinfection is a major mean to control and eliminate pathogenic bacteria, particularly those with multidrug resistance (MDR phenotype. However, exposition to biocides results in an adaptive response in microorganisms, causing them to display a wide range of resistance mechanisms. Numerous microorganisms are characterized by either natural resistance to chemical compounds or an ability to adapt to biocides using various strategies, such as: modification of cell surface structures (lipopolisaccharide, membrane fatty acids, over-expression of efflux pumps (a system for active transport of toxic compounds out of bacterial cell, enzymatic inactivation of biocides or altering biocide targets. For instance, it was shown that in vitro exposition of Salmonella Typhimurium to subinhibitory concentration of biocides (triclosan, quaternary ammonium compounds [QACs] resulted in selection of variants resistant to tested biocides and, additionally, to acridine dyes and antibiotics. Bacillus subtilis and Micrococcus luteus strains isolated from chlorine dioxide containing disinfection devices were found to be resistant to chlorine dioxide and also to other oxidizing compounds, such as peracetic acid and hydrogen peroxide. Interaction between chemical compounds, including disinfectants and microbial cells, can create a serious threat to public health and sanitary-hygienic security. This phenomenon is connected with factor risk that intensify the probability of selection and dissemination of multidrug resistance among pathogenic bacteria.

  10. Bacterial cell numbers and community structures of seawater biofilms depend on the attachment substratum

    KAUST Repository

    Yap, Scott A.

    2018-02-02

    Seawater is increasingly being used as a source for various industrial applications. For such applications, biofilm growth creates various problems including but not limited to pipe biocorrosion. In this study, it is hypothesized that the material type is preferred by certain bacterial populations in the seawater to attach and establish biofilms. By comparing differences in the total cell counts and microbial communities attached to high-density polyethylene (HDPE), polycarbonate, stainless steel (SS316) and titanium, the appropriate material can be used to minimize biofilm growth. All four materials have hydrophilic surfaces, but polycarbonate exhibits higher surface roughness. There were no significant differences in the cell numbers attached to polycarbonate, HDPE and titanium. Instead, there were significantly fewer cells attached to SS316. However, there was a higher relative abundance of genera associated with opportunistic pathogens on SS316. Copy numbers of genes representing Desulfobacteraceae and Desulfobulbaceae, both of which are sulfate-reducing bacteria (SRB), were approximately 10-fold higher in biofilms sampled from SS316. The enrichment of SRB in the biofilm associated with SS316 indicates that this material may be prone to biocorrosion. This study highlights the need for industries to consider the choice of material used in seawater applications to minimize microbial-associated problems.

  11. Phenotypic T cell exhaustion in a murine model of bacterial infection in the setting of pre-existing malignancy.

    Directory of Open Access Journals (Sweden)

    Rohit Mittal

    Full Text Available While much of cancer immunology research has focused on anti-tumor immunity both systemically and within the tumor microenvironment, little is known about the impact of pre-existing malignancy on pathogen-specific immune responses. Here, we sought to characterize the antigen-specific CD8+ T cell response following a bacterial infection in the setting of pre-existing pancreatic adenocarcinoma. Mice with established subcutaneous pancreatic adenocarcinomas were infected with Listeria monocytogenes, and antigen-specific CD8+ T cell responses were compared to those in control mice without cancer. While the kinetics and magnitude of antigen-specific CD8+ T cell expansion and accumulation was comparable between the cancer and non-cancer groups, bacterial antigen-specific CD8+ T cells and total CD4+ and CD8+ T cells in cancer mice exhibited increased expression of the coinhibitory receptors BTLA, PD-1, and 2B4. Furthermore, increased inhibitory receptor expression was associated with reduced IFN-γ and increased IL-2 production by bacterial antigen-specific CD8+ T cells in the cancer group. Taken together, these data suggest that cancer's immune suppressive effects are not limited to the tumor microenvironment, but that pre-existing malignancy induces phenotypic exhaustion in T cells by increasing expression of coinhibitory receptors and may impair pathogen-specific CD8+ T cell functionality and differentiation.

  12. Sensing the Structural Differences in Cellulose from Apple and Bacterial Cell Wall Materials by Raman and FT-IR Spectroscopy

    Science.gov (United States)

    Szymańska-Chargot, Monika; Cybulska, Justyna; Zdunek, Artur

    2011-01-01

    Raman and Fourier Transform Infrared (FT-IR) spectroscopy was used for assessment of structural differences of celluloses of various origins. Investigated celluloses were: bacterial celluloses cultured in presence of pectin and/or xyloglucan, as well as commercial celluloses and cellulose extracted from apple parenchyma. FT-IR spectra were used to estimate of the Iβ content, whereas Raman spectra were used to evaluate the degree of crystallinity of the cellulose. The crystallinity index (XCRAMAN%) varied from −25% for apple cellulose to 53% for microcrystalline commercial cellulose. Considering bacterial cellulose, addition of xyloglucan has an impact on the percentage content of cellulose Iβ. However, addition of only xyloglucan or only pectins to pure bacterial cellulose both resulted in a slight decrease of crystallinity. However, culturing bacterial cellulose in the presence of mixtures of xyloglucan and pectins results in an increase of crystallinity. The results confirmed that the higher degree of crystallinity, the broader the peak around 913 cm−1. Among all bacterial celluloses the bacterial cellulose cultured in presence of xyloglucan and pectin (BCPX) has the most similar structure to those observed in natural primary cell walls. PMID:22163913

  13. Quorum sensing integrates environmental cues, cell density and cell history to control bacterial competence

    NARCIS (Netherlands)

    Moreno-Gámez, Stefany; Sorg, Robin A; Domenech, Arnau; Kjos, Morten; Weissing, Franz J; van Doorn, G Sander; Veening, Jan-Willem

    2017-01-01

    Streptococcus pneumoniae becomes competent for genetic transformation when exposed to an autoinducer peptide known as competence-stimulating peptide (CSP). This peptide was originally described as a quorum-sensing signal, enabling individual cells to regulate competence in response to population

  14. Structure of the complex between teicoplanin and a bacterial cell-wall peptide: use of a carrier-protein approach

    Energy Technology Data Exchange (ETDEWEB)

    Economou, Nicoleta J.; Zentner, Isaac J. [Drexel University College of Medicine, 245 North 15th Street, Philadelphia, PA 19102 (United States); Lazo, Edwin; Jakoncic, Jean; Stojanoff, Vivian [Brookhaven National Laboratory, Upton, NY 11973 (United States); Weeks, Stephen D.; Grasty, Kimberly C.; Cocklin, Simon; Loll, Patrick J. [Drexel University College of Medicine, 245 North 15th Street, Philadelphia, PA 19102 (United States)

    2013-04-01

    Using a carrier-protein strategy, the structure of teicoplanin bound to its bacterial cell-wall target has been determined. The structure reveals the molecular determinants of target recognition, flexibility in the antibiotic backbone and intrinsic radiation sensitivity of teicoplanin. Multidrug-resistant bacterial infections are commonly treated with glycopeptide antibiotics such as teicoplanin. This drug inhibits bacterial cell-wall biosynthesis by binding and sequestering a cell-wall precursor: a d-alanine-containing peptide. A carrier-protein strategy was used to crystallize the complex of teicoplanin and its target peptide by fusing the cell-wall peptide to either MBP or ubiquitin via native chemical ligation and subsequently crystallizing the protein–peptide–antibiotic complex. The 2.05 Å resolution MBP–peptide–teicoplanin structure shows that teicoplanin recognizes its ligand through a combination of five hydrogen bonds and multiple van der Waals interactions. Comparison of this teicoplanin structure with that of unliganded teicoplanin reveals a flexibility in the antibiotic peptide backbone that has significant implications for ligand recognition. Diffraction experiments revealed an X-ray-induced dechlorination of the sixth amino acid of the antibiotic; it is shown that teicoplanin is significantly more radiation-sensitive than other similar antibiotics and that ligand binding increases radiosensitivity. Insights derived from this new teicoplanin structure may contribute to the development of next-generation antibacterials designed to overcome bacterial resistance.

  15. Structure of the complex between teicoplanin and a bacterial cell-wall peptide: use of a carrier-protein approach

    International Nuclear Information System (INIS)

    Economou, Nicoleta J.; Zentner, Isaac J.; Lazo, Edwin; Jakoncic, Jean; Stojanoff, Vivian; Weeks, Stephen D.; Grasty, Kimberly C.; Cocklin, Simon; Loll, Patrick J.

    2013-01-01

    Using a carrier-protein strategy, the structure of teicoplanin bound to its bacterial cell-wall target has been determined. The structure reveals the molecular determinants of target recognition, flexibility in the antibiotic backbone and intrinsic radiation sensitivity of teicoplanin. Multidrug-resistant bacterial infections are commonly treated with glycopeptide antibiotics such as teicoplanin. This drug inhibits bacterial cell-wall biosynthesis by binding and sequestering a cell-wall precursor: a d-alanine-containing peptide. A carrier-protein strategy was used to crystallize the complex of teicoplanin and its target peptide by fusing the cell-wall peptide to either MBP or ubiquitin via native chemical ligation and subsequently crystallizing the protein–peptide–antibiotic complex. The 2.05 Å resolution MBP–peptide–teicoplanin structure shows that teicoplanin recognizes its ligand through a combination of five hydrogen bonds and multiple van der Waals interactions. Comparison of this teicoplanin structure with that of unliganded teicoplanin reveals a flexibility in the antibiotic peptide backbone that has significant implications for ligand recognition. Diffraction experiments revealed an X-ray-induced dechlorination of the sixth amino acid of the antibiotic; it is shown that teicoplanin is significantly more radiation-sensitive than other similar antibiotics and that ligand binding increases radiosensitivity. Insights derived from this new teicoplanin structure may contribute to the development of next-generation antibacterials designed to overcome bacterial resistance

  16. Short-term variability in bacterial abundance, cell properties, and incorporation of leucine and thymidine in subarctic sea ice

    DEFF Research Database (Denmark)

    Kaartokallio, H.; Søgaard, D.H.; Norman, L.

    2013-01-01

    Sea ice is a biome of immense size and provides a range of habitats for diverse microbial communities, many of which are adapted to living at low temperatures and high salinities in brines. We measured simultaneous incorporation of thymidine (TdR) and leucine (Leu), bacterial cell abundance...

  17. Ascites bacterial burden and immune cell profile are associated with poor clinical outcomes in the absence of overt infection.

    Directory of Open Access Journals (Sweden)

    Kevin J Fagan

    Full Text Available Bacterial infections, most commonly spontaneous bacterial peritonitis in patients with ascites, occur in one third of admitted patients with cirrhosis, and account for a 4-fold increase in mortality. Bacteria are isolated from less than 40% of ascites infections by culture, necessitating empirical antibiotic treatment, but culture-independent studies suggest bacteria are commonly present, even in the absence of overt infection. Widespread detection of low levels of bacteria in ascites, in the absence of peritonitis, suggests immune impairment may contribute to higher susceptibility to infection in cirrhotic patients. However, little is known about the role of ascites leukocyte composition and function in this context. We determined ascites bacterial composition by quantitative PCR and 16S rRNA gene sequencing in 25 patients with culture-negative, non-neutrocytic ascites, and compared microbiological data with ascites and peripheral blood leukocyte composition and phenotype. Bacterial DNA was detected in ascitic fluid from 23 of 25 patients, with significant positive correlations between bacterial DNA levels and poor 6-month clinical outcomes (death, readmission. Ascites leukocyte composition was variable, but dominated by macrophages or T lymphocytes, with lower numbers of B lymphocytes and natural killer cells. Consistent with the hypothesis that impaired innate immunity contributes to susceptibility to infection, high bacterial DNA burden was associated with reduced major histocompatibility complex class II expression on ascites (but not peripheral blood monocytes/macrophages. These data indicate an association between the presence of ascites bacterial DNA and early death and readmission in patients with decompensated cirrhosis. They further suggest that impairment of innate immunity contributes to increased bacterial translocation, risk of peritonitis, or both.

  18. Connecting the dots of the bacterial cell cycle: Coordinating chromosome replication and segregation with cell division.

    Science.gov (United States)

    Hajduk, Isabella V; Rodrigues, Christopher D A; Harry, Elizabeth J

    2016-05-01

    Proper division site selection is crucial for the survival of all organisms. What still eludes us is how bacteria position their division site with high precision, and in tight coordination with chromosome replication and segregation. Until recently, the general belief, at least in the model organisms Bacillus subtilis and Escherichia coli, was that spatial regulation of division comes about by the combined negative regulatory mechanisms of the Min system and nucleoid occlusion. However, as we review here, these two systems cannot be solely responsible for division site selection and we highlight additional regulatory mechanisms that are at play. In this review, we put forward evidence of how chromosome replication and segregation may have direct links with cell division in these bacteria and the benefit of recent advances in chromosome conformation capture techniques in providing important information about how these three processes mechanistically work together to achieve accurate generation of progenitor cells. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  19. Bacterial Ghosts of Escherichia coli Drive Efficient Maturation of Bovine Monocyte-Derived Dendritic Cells.

    Directory of Open Access Journals (Sweden)

    Irshad Ahmed Hajam

    Full Text Available Bacterial ghosts (BGs are empty cell envelopes derived from Gram-negative bacteria. They not only represent a potential platform for development of novel vaccines but also provide a tool for efficient adjuvant and antigen delivery system. In the present study, we investigated the interaction between BGs of Escherichia coli (E. coli and bovine monocyte-derived dendritic cells (MoDCs. MoDCs are highly potent antigen-presenting cells and have the potential to act as a powerful tool for manipulating the immune system. We generated bovine MoDCs in vitro from blood monocytes using E. coli expressed bovine GM-CSF and IL-4 cytokines. These MoDCs displayed typical morphology and functions similar to DCs. We further investigated the E. coli BGs to induce maturation of bovine MoDCs in comparison to E. coli lipopolysaccharide (LPS. We observed the maturation marker molecules such as MHC-II, CD80 and CD86 were induced early and at higher levels in BG stimulated MoDCs as compared to the LPS stimulated MoDCs. BG mediated stimulation induced significantly higher levels of cytokine expression in bovine MoDCs than LPS. Both pro-inflammatory (IL-12 and TNF-α and anti-inflammatory (IL-10 cytokines were induced in MoDCs after BGs stimulation. We further analysed the effects of BGs on the bovine MoDCs in an allogenic mixed lymphocyte reaction (MLR. We found the BG-treated bovine MoDCs had significantly (p<0.05 higher capacity to stimulate allogenic T cell proliferation in MLR as compared to the LPS. Taken together, these findings demonstrate the E. coli BGs induce a strong activation and maturation of bovine MoDCs.

  20. Genome-wide dynamics of a bacterial response to antibiotics that target the cell envelope

    Science.gov (United States)

    2011-01-01

    Background A decline in the discovery of new antibacterial drugs, coupled with a persistent rise in the occurrence of drug-resistant bacteria, has highlighted antibiotics as a diminishing resource. The future development of new drugs with novel antibacterial activities requires a detailed understanding of adaptive responses to existing compounds. This study uses Streptomyces coelicolor A3(2) as a model system to determine the genome-wide transcriptional response following exposure to three antibiotics (vancomycin, moenomycin A and bacitracin) that target distinct stages of cell wall biosynthesis. Results A generalised response to all three antibiotics was identified which involves activation of transcription of the cell envelope stress sigma factor σE, together with elements of the stringent response, and of the heat, osmotic and oxidative stress regulons. Attenuation of this system by deletion of genes encoding the osmotic stress sigma factor σB or the ppGpp synthetase RelA reduced resistance to both vancomycin and bacitracin. Many antibiotic-specific transcriptional changes were identified, representing cellular processes potentially important for tolerance to each antibiotic. Sensitivity studies using mutants constructed on the basis of the transcriptome profiling confirmed a role for several such genes in antibiotic resistance, validating the usefulness of the approach. Conclusions Antibiotic inhibition of bacterial cell wall biosynthesis induces both common and compound-specific transcriptional responses. Both can be exploited to increase antibiotic susceptibility. Regulatory networks known to govern responses to environmental and nutritional stresses are also at the core of the common antibiotic response, and likely help cells survive until any specific resistance mechanisms are fully functional. PMID:21569315

  1. Genome-wide dynamics of a bacterial response to antibiotics that target the cell envelope

    Directory of Open Access Journals (Sweden)

    Tran Ngat

    2011-05-01

    Full Text Available Abstract Background A decline in the discovery of new antibacterial drugs, coupled with a persistent rise in the occurrence of drug-resistant bacteria, has highlighted antibiotics as a diminishing resource. The future development of new drugs with novel antibacterial activities requires a detailed understanding of adaptive responses to existing compounds. This study uses Streptomyces coelicolor A3(2 as a model system to determine the genome-wide transcriptional response following exposure to three antibiotics (vancomycin, moenomycin A and bacitracin that target distinct stages of cell wall biosynthesis. Results A generalised response to all three antibiotics was identified which involves activation of transcription of the cell envelope stress sigma factor σE, together with elements of the stringent response, and of the heat, osmotic and oxidative stress regulons. Attenuation of this system by deletion of genes encoding the osmotic stress sigma factor σB or the ppGpp synthetase RelA reduced resistance to both vancomycin and bacitracin. Many antibiotic-specific transcriptional changes were identified, representing cellular processes potentially important for tolerance to each antibiotic. Sensitivity studies using mutants constructed on the basis of the transcriptome profiling confirmed a role for several such genes in antibiotic resistance, validating the usefulness of the approach. Conclusions Antibiotic inhibition of bacterial cell wall biosynthesis induces both common and compound-specific transcriptional responses. Both can be exploited to increase antibiotic susceptibility. Regulatory networks known to govern responses to environmental and nutritional stresses are also at the core of the common antibiotic response, and likely help cells survive until any specific resistance mechanisms are fully functional.

  2. X-ray crystallography and its impact on understanding bacterial cell wall remodeling processes.

    Science.gov (United States)

    Büttner, Felix Michael; Renner-Schneck, Michaela; Stehle, Thilo

    2015-02-01

    The molecular structure of matter defines its properties and function. This is especially true for biological macromolecules such as proteins, which participate in virtually all biochemical processes. A three dimensional structural model of a protein is thus essential for the detailed understanding of its physiological function and the characterization of essential properties such as ligand binding and reaction mechanism. X-ray crystallography is a well-established technique that has been used for many years, but it is still by far the most widely used method for structure determination. A particular strength of this technique is the elucidation of atomic details of molecular interactions, thus providing an invaluable tool for a multitude of scientific projects ranging from the structural classification of macromolecules over the validation of enzymatic mechanisms or the understanding of host-pathogen interactions to structure-guided drug design. In the first part of this review, we describe essential methodological and practical aspects of X-ray crystallography. We provide some pointers that should allow researchers without a background in structural biology to assess the overall quality and reliability of a crystal structure. To highlight its potential, we then survey the impact X-ray crystallography has had on advancing an understanding of a class of enzymes that modify the bacterial cell wall. A substantial number of different bacterial amidase structures have been solved, mostly by X-ray crystallography. Comparison of these structures highlights conserved as well as divergent features. In combination with functional analyses, structural information on these enzymes has therefore proven to be a valuable template not only for understanding their mechanism of catalysis, but also for targeted interference with substrate binding. Copyright © 2015 Elsevier GmbH. All rights reserved.

  3. Phospholipase D promotes Arcanobacterium haemolyticum adhesion via lipid raft remodeling and host cell death following bacterial invasion

    Directory of Open Access Journals (Sweden)

    Carlson Petteri

    2010-10-01

    Full Text Available Abstract Background Arcanobacterium haemolyticum is an emerging bacterial pathogen, causing pharyngitis and more invasive infections. This organism expresses an unusual phospholipase D (PLD, which we propose promotes bacterial pathogenesis through its action on host cell membranes. The pld gene is found on a genomic region of reduced %G + C, suggesting recent horizontal acquisition. Results Recombinant PLD rearranged HeLa cell lipid rafts in a dose-dependent manner and this was inhibited by cholesterol sequestration. PLD also promoted host cell adhesion, as a pld mutant had a 60.3% reduction in its ability to adhere to HeLa cells as compared to the wild type. Conversely, the pld mutant appeared to invade HeLa cells approximately two-fold more efficiently as the wild type. This finding was attributable to a significant loss of host cell viability following secretion of PLD from intracellular bacteria. As determined by viability assay, only 15.6% and 82.3% of HeLa cells remained viable following invasion by the wild type or pld mutant, respectively, as compared to untreated HeLa cells. Transmission electron microscopy of HeLa cells inoculated with A. haemolyticum strains revealed that the pld mutant was contained within intracellular vacuoles, as compared to the wild type, which escaped the vacuole. Wild type-infected HeLa cells also displayed the hallmarks of necrosis. Similarly inoculated HeLa cells displayed no signs of apoptosis, as measured by induction of caspase 3/7, 8 or 9 activities. Conclusions These data indicate that PLD enhances bacterial adhesion and promotes host cell necrosis following invasion, and therefore, may be important in the disease pathogenesis of A. haemolyticum infections.

  4. The Membrane Steps of Bacterial Cell Wall Synthesis as Antibiotic Targets

    Directory of Open Access Journals (Sweden)

    Yao Liu

    2016-08-01

    Full Text Available Peptidoglycan is the major component of the cell envelope of virtually all bacteria. It has structural roles and acts as a selective sieve for molecules from the outer environment. Peptidoglycan synthesis is therefore one of the most important biogenesis pathways in bacteria and has been studied extensively over the last twenty years. The pathway starts in the cytoplasm, continues in the cytoplasmic membrane and finishes in the periplasmic space, where the precursor is polymerized into the peptidoglycan layer. A number of proteins involved in this pathway, such as the Mur enzymes and the penicillin binding proteins (PBPs, have been studied and regarded as good targets for antibiotics. The present review focuses on the membrane steps of peptidoglycan synthesis that involve two enzymes, MraY and MurG, the inhibitors of these enzymes and the inhibition mechanisms. We also discuss the challenges of targeting these two cytoplasmic membrane (associated proteins in bacterial cells and the perspectives on how to overcome the issues.

  5. Altered host cell-bacteria interaction due to nanoparticle interaction with a bacterial biofilm.

    Science.gov (United States)

    Raftery, Tara D; Lindler, Heidi; McNealy, Tamara L

    2013-02-01

    Nanoparticle (NP) use in everyday applications creates the potential for NPs to enter the environment where, in aquatic systems, they are likely to settle on substrates and interact with microbial communities. Legionella pneumophila biofilms are found as part of microbial communities in both natural and man-made environments, especially in man-made cooling systems. The bacterium is the causative agent of Legionnaires' disease. Legionella requires a host cell for replication in the environment, and amoebae commonly serve as this host cell. Our previous work demonstrated significant changes in Legionella biofilm morphology after exposure to 0.7 μg/L gold NPs (AuNPs). Here, we investigate how these morphology changes alter host-bacteria interactions using Acanthamoeba polyphaga as a model. Host-bacteria-NP interactions are affected by NP characteristics. Biofilms exposed to 4- and 18-nm, citrate-capped, spherical AuNPs significantly altered the grazing ability of A. polyphaga, which was not observed in biofilms exposed to 24-nm polystyrene beads. Uptake and replication of NP-exposed planktonic L. pneumophila within A. polyphaga were not altered regardless of NP size or core chemistry. Nanomaterial effects on the interaction of benthic organisms and bacteria may be directly or, as shown here, indirectly dependent on bacterial morphology. NP contamination therefore may alter interactions in a normal ecosystem function.

  6. Biosynthesis of Bacterial Cellulose/Carboxylic Multi-Walled Carbon Nanotubes for Enzymatic Biofuel Cell Application

    Directory of Open Access Journals (Sweden)

    Pengfei Lv

    2016-03-01

    Full Text Available Novel nanocomposites comprised of bacterial cellulose (BC with carboxylic multi-walled carbon nanotubes (c-MWCNTs incorporated into the BC matrix were prepared through a simple method of biosynthesis. The biocathode and bioanode for the enzyme biological fuel cell (EBFC were prepared using BC/c-MWCNTs composite injected by laccase (Lac and glucose oxidase (GOD with the aid of glutaraldehyde (GA crosslinking. Biosynthesis of BC/c-MWCNTs composite was characterized by digital photos, scanning electron microscope (SEM, and Fourier Transform Infrared (FTIR. The experimental results indicated the successful incorporation of c-MWCNTs into the BC. The electrochemical and biofuel performance were evaluated by cyclic voltammetry (CV and linear sweep voltammetry (LSV. The power density and current density of EBFCs were recorded at 32.98 µW/cm3 and 0.29 mA/cm3, respectively. Additionally, the EBFCs also showed acceptable stability. Preliminary tests on double cells indicated that renewable BC have great potential in the application field of EBFCs.

  7. Bacterial surface appendages strongly impact nanomechanical and electrokinetic properties of Escherichia coli cells subjected to osmotic stress.

    Directory of Open Access Journals (Sweden)

    Grégory Francius

    Full Text Available The physicochemical properties and dynamics of bacterial envelope, play a major role in bacterial activity. In this study, the morphological, nanomechanical and electrohydrodynamic properties of Escherichia coli K-12 mutant cells were thoroughly investigated as a function of bulk medium ionic strength using atomic force microscopy (AFM and electrokinetics (electrophoresis. Bacteria were differing according to genetic alterations controlling the production of different surface appendages (short and rigid Ag43 adhesins, longer and more flexible type 1 fimbriae and F pilus. From the analysis of the spatially resolved force curves, it is shown that cells elasticity and turgor pressure are not only depending on bulk salt concentration but also on the presence/absence and nature of surface appendage. In 1 mM KNO(3, cells without appendages or cells surrounded by Ag43 exhibit large Young moduli and turgor pressures (∼700-900 kPa and ∼100-300 kPa respectively. Under similar ionic strength condition, a dramatic ∼50% to ∼70% decrease of these nanomechanical parameters was evidenced for cells with appendages. Qualitatively, such dependence of nanomechanical behavior on surface organization remains when increasing medium salt content to 100 mM, even though, quantitatively, differences are marked to a much smaller extent. Additionally, for a given surface appendage, the magnitude of the nanomechanical parameters decreases significantly when increasing bulk salt concentration. This effect is ascribed to a bacterial exoosmotic water loss resulting in a combined contraction of bacterial cytoplasm together with an electrostatically-driven shrinkage of the surface appendages. The former process is demonstrated upon AFM analysis, while the latter, inaccessible upon AFM imaging, is inferred from electrophoretic data interpreted according to advanced soft particle electrokinetic theory. Altogether, AFM and electrokinetic results clearly demonstrate the

  8. Bacterial biofilm mechanical properties persist upon antibiotic treatment and survive cell death

    International Nuclear Information System (INIS)

    Zrelli, K; Galy, O; Henry, N; Latour-Lambert, P; Ghigo, J M; Beloin, C; Kirwan, L

    2013-01-01

    Bacteria living on surfaces form heterogeneous three-dimensional consortia known as biofilms, where they exhibit many specific properties one of which is an increased tolerance to antibiotics. Biofilms are maintained by a polymeric network and display physical properties similar to that of complex fluids. In this work, we address the question of the impact of antibiotic treatment on the physical properties of biofilms based on recently developed tools enabling the in situ mapping of biofilm local mechanical properties at the micron scale. This approach takes into account the material heterogeneity and reveals the spatial distribution of all the small changes that may occur in the structure. With an Escherichia coli biofilm, we demonstrate using in situ fluorescent labeling that the two antibiotics ofloxacin and ticarcillin—targeting DNA replication and membrane assembly, respectively—induced no detectable alteration of the biofilm mechanical properties while they killed the vast majority of the cells. In parallel, we show that a proteolytic enzyme that cleaves extracellular proteins into short peptides, but does not alter bacterial viability in the biofilm, clearly affects the mechanical properties of the biofilm structure, inducing a significant increase of the material compliance. We conclude that conventional biofilm control strategy relying on the use of biocides targeting cells is missing a key target since biofilm structural integrity is preserved. This is expected to efficiently promote biofilm resilience, especially in the presence of persister cells. In contrast, the targeting of polymer network cross-links—among which extracellular proteins emerge as major players—offers a promising route for the development of rational multi-target strategies to fight against biofilms. (paper)

  9. Bacterial Ghosts of Escherichia coli Drive Efficient Maturation of Bovine Monocyte-Derived Dendritic Cells.

    Science.gov (United States)

    Hajam, Irshad Ahmed; Dar, Pervaiz Ahmad; Appavoo, Elamurugan; Kishore, Subodh; Bhanuprakash, Veerakyathappa; Ganesh, Kondabattula

    2015-01-01

    Bacterial ghosts (BGs) are empty cell envelopes derived from Gram-negative bacteria. They not only represent a potential platform for development of novel vaccines but also provide a tool for efficient adjuvant and antigen delivery system. In the present study, we investigated the interaction between BGs of Escherichia coli (E. coli) and bovine monocyte-derived dendritic cells (MoDCs). MoDCs are highly potent antigen-presenting cells and have the potential to act as a powerful tool for manipulating the immune system. We generated bovine MoDCs in vitro from blood monocytes using E. coli expressed bovine GM-CSF and IL-4 cytokines. These MoDCs displayed typical morphology and functions similar to DCs. We further investigated the E. coli BGs to induce maturation of bovine MoDCs in comparison to E. coli lipopolysaccharide (LPS). We observed the maturation marker molecules such as MHC-II, CD80 and CD86 were induced early and at higher levels in BG stimulated MoDCs as compared to the LPS stimulated MoDCs. BG mediated stimulation induced significantly higher levels of cytokine expression in bovine MoDCs than LPS. Both pro-inflammatory (IL-12 and TNF-α) and anti-inflammatory (IL-10) cytokines were induced in MoDCs after BGs stimulation. We further analysed the effects of BGs on the bovine MoDCs in an allogenic mixed lymphocyte reaction (MLR). We found the BG-treated bovine MoDCs had significantly (pBGs induce a strong activation and maturation of bovine MoDCs.

  10. In vitro behaviors of rat mesenchymal stem cells on bacterial celluloses with different moduli

    Energy Technology Data Exchange (ETDEWEB)

    Taokaew, Siriporn [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330 (Thailand); Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH 44325-3906 (United States); Phisalaphong, Muenduen [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330 (Thailand); Zhang Newby, Bi-min, E-mail: bimin@uakron.edu [Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH 44325-3906 (United States)

    2014-05-01

    Compressive moduli of bacteria-synthesized cellulose (BC) were altered by two drying techniques: ambient-air drying and freeze drying. While no significant differences in dry weight were found, their cross-sectional structures and thickness varied greatly. Freeze dried BCs had loose cross-sectional structures and a thickness of ∼ 4.7 mm, whereas air dried BCs had more compacted cross-sectional structures and a thickness of ∼ 0.1 mm. The compressive moduli of the rehydrated freeze dried and rehydrated air dried BCs were measured to be 21.06 ± 0.22 kPa and 90.09 ± 21.07 kPa, respectively. When rat mesenchymal stem cells (rMSCs) were seeded on these BCs, they maintained a round morphology in the first 3 days of cultivation. More spread-out morphology and considerable proliferation on freeze dried BCs were observed in 7 days, but not on air-dried BCs. The cells were further grown for 3 weeks in the absence and presence of differentiation agents. Without using any differentiation agents, no detectable differentiation was noticed for rMSCs further cultivated on both types of BC. With differentiation inducing agents, chondrogenic differentiation, visualized by histological staining, was observed in some area of the rehydrated freeze dried BCs; while osteogenic differentiation was noticed on the stiffer rehydrated air dried BCs. - Graphical abstract: In the presence of induction agents, rat mesenchymal stem cells (rMSCs) preferentially differentiated into osteocytes on stiffer air dried BC films. - Highlights: • Bacterial cellulose (BC) sheets with different moduli generated by drying differently • Air-dried BC exhibited a modulus similar to that of bone. • Freeze-dried BC showed a modulus in the range of that of muscle. • Air-dried BC promoted the differentiation of rMSCs into osteocytes. • Freeze-dried BC promoted the differentiation of rMSCs into chondrocytes.

  11. Studies on penetration of antibiotic in bacterial cells in space conditions (7-IML-1)

    Science.gov (United States)

    Tixador, R.

    1992-01-01

    The Cytos 2 experiment was performed aboard Salyut 7 in order to test the antibiotic sensitivity of bacteria cultivated in vitro in space. An increase of the Minimal Inhibitory Concentration (MIC) in the inflight cultures (i.e., an increase of the antibiotic resistance) was observed. Complementary studies of the ultrastructure showed a thickening of the cell envelope. In order to confirm the results of the Cytos 2 experiment, we performed the ANTIBIO experiment during the D1 mission to try to differentiate, by means of the 1 g centrifuge in the Biorack, between the biological effects of cosmic rays and those caused by microgravity conditions. The originality of this experiment was in the fact that it was designed to test the antibiotic sensitivity of bacteria cultivated in vitro during the orbital phase of the flight. The results show an increase in resistance to Colistin in in-flight bacteria. The MIC is practically double in the in-flight cultures. A cell count of living bacteria in the cultures containing the different Colistin concentrations showed a significant difference between the cultures developed during space flight and the ground based cultures. The comparison between the 1 g and 0 g in-flight cultures show similar behavior for the two sets. Nevertheless, a small difference between the two sets of ground based control cultures was noted. The cultures developed on the ground centrifuge (1.4 g) present a slight decrease in comparison with the cultures developed in the static rack (1 g). In order to approach the mechanisms of the increase of antibiotic resistance on bacteria cultivated in vitro in space, we have proposed the study on penetration of antibiotics in bacterial cells in space conditions. This experiment was selected for the International Microgravity Laboratory 1 (IML-1) mission.

  12. Bacterial biofilm mechanical properties persist upon antibiotic treatment and survive cell death

    Science.gov (United States)

    Zrelli, K.; Galy, O.; Latour-Lambert, P.; Kirwan, L.; Ghigo, J. M.; Beloin, C.; Henry, N.

    2013-12-01

    Bacteria living on surfaces form heterogeneous three-dimensional consortia known as biofilms, where they exhibit many specific properties one of which is an increased tolerance to antibiotics. Biofilms are maintained by a polymeric network and display physical properties similar to that of complex fluids. In this work, we address the question of the impact of antibiotic treatment on the physical properties of biofilms based on recently developed tools enabling the in situ mapping of biofilm local mechanical properties at the micron scale. This approach takes into account the material heterogeneity and reveals the spatial distribution of all the small changes that may occur in the structure. With an Escherichia coli biofilm, we demonstrate using in situ fluorescent labeling that the two antibiotics ofloxacin and ticarcillin—targeting DNA replication and membrane assembly, respectively—induced no detectable alteration of the biofilm mechanical properties while they killed the vast majority of the cells. In parallel, we show that a proteolytic enzyme that cleaves extracellular proteins into short peptides, but does not alter bacterial viability in the biofilm, clearly affects the mechanical properties of the biofilm structure, inducing a significant increase of the material compliance. We conclude that conventional biofilm control strategy relying on the use of biocides targeting cells is missing a key target since biofilm structural integrity is preserved. This is expected to efficiently promote biofilm resilience, especially in the presence of persister cells. In contrast, the targeting of polymer network cross-links—among which extracellular proteins emerge as major players—offers a promising route for the development of rational multi-target strategies to fight against biofilms.

  13. Bacterial stress

    Indian Academy of Sciences (India)

    First page Back Continue Last page Graphics. Bacterial stress. Physicochemical and chemical parameters: temperature, pressure, pH, salt concentration, oxygen, irradiation. Nutritional depravation: nutrient starvation, water shortage. Toxic compounds: Antibiotics, heavy metals, toxins, mutagens. Interactions with other cells: ...

  14. To Eat and to Be Eaten: Mutual Metabolic Adaptations of Immune Cells and Intracellular Bacterial Pathogens upon Infection

    Science.gov (United States)

    Eisenreich, Wolfgang; Rudel, Thomas; Heesemann, Jürgen; Goebel, Werner

    2017-01-01

    Intracellular bacterial pathogens (IBPs) invade and replicate in different cell types including immune cells, in particular of the innate immune system (IIS) during infection in the acute phase. However, immune cells primarily function as essential players in the highly effective and integrated host defense systems comprising the IIS and the adaptive immune system (AIS), which cooperatively protect the host against invading microbes including IBPs. As countermeasures, the bacterial pathogens (and in particular the IBPs) have developed strategies to evade or reprogram the IIS at various steps. The intracellular replication capacity and the anti-immune defense responses of the IBP's as well as the specific antimicrobial responses of the immune cells of the innate and the AIS depend on specific metabolic programs of the IBPs and their host cells. The metabolic programs of the immune cells supporting or counteracting replication of the IBPs appear to be mutually exclusive. Indeed, recent studies show that upon interaction of naïve, metabolically quiescent immune cells with IBPs, different metabolic activation processes occur which may result in the provision of a survival and replication niche for the pathogen or its eradication. It is therefore likely that within a possible host cell population subsets exist that are metabolically programmed for pro- or anti-microbial conditions. These metabolic programs may be triggered by the interactions between different bacterial agonistic components and host cell receptors. In this review, we summarize the current status in the field and discuss metabolic adaptation processes within immune cells of the IIS and the IBPs that support or restrict the intracellular replication of the pathogens. PMID:28752080

  15. Role of Sulfhydryl Sites on Bacterial Cell Walls in the Biosorption, Mobility and Bioavailability of Mercury and Uranium

    Energy Technology Data Exchange (ETDEWEB)

    Myneni, Satish C. B. [Princeton Univ., NJ (United States). Dept. of Geosciences; Fein, Jeremy [Univ. of Notre Dame, IN (United States). Dept. of Civil Engineering and Geological Sciences; Mishra, Bhoopesh [Argonne National Lab. (ANL), Argonne, IL (United States)

    2016-09-16

    Bacteria are ubiquitous in a wide-range of low temperature aqueous systems, and can strongly affect the distribution and transport of metals and radionuclides in the environment. However, the role of metal adsorption onto bacteria, via the reactive cell wall functional groups, has been largely overlooked. Previous macroscale metal sorption, and XAS studies have shown that carboxyl and phosphoryl functional groups to be the important metal binding groups on bacterial cell walls and the sulfhydryl groups were not considered. The goal of our investigation was to evaluate the density of the sulfhydryl sites on different bacterial cell membranes that are common to soil systems, the binding affinities of these reactive groups towards Hg, and how this binding modifies the speciation of Hg in the natural waters.

  16. Exposure of Bacterial Biofilms to Electrical Current Leads to Cell Death Mediated in Part by Reactive Oxygen Species.

    Science.gov (United States)

    Brinkman, Cassandra L; Schmidt-Malan, Suzannah M; Karau, Melissa J; Greenwood-Quaintance, Kerryl; Hassett, Daniel J; Mandrekar, Jayawant N; Patel, Robin

    2016-01-01

    Bacterial biofilms may form on indwelling medical devices such as prosthetic joints, heart valves and catheters, causing challenging-to-treat infections. We have previously described the 'electricidal effect', in which bacterial biofilms are decreased following exposure to direct electrical current. Herein, we sought to determine if the decreased bacterial quantities are due to detachment of biofilms or cell death and to investigate the role that reactive oxygen species (ROS) play in the observed effect. Using confocal and electron microscopy and flow cytometry, we found that direct current (DC) leads to cell death and changes in the architecture of biofilms formed by Gram-positive and Gram-negative bacteria. Reactive oxygen species (ROS) appear to play a role in DC-associated cell death, as there was an increase in ROS-production by Staphylococcus aureus and Staphylococcus epidermidis biofilms following exposure to DC. An increase in the production of ROS response enzymes catalase and superoxide dismutase (SOD) was observed for S. aureus, S. epidermidis and Pseudomonas aeruginosa biofilms following exposure to DC. Additionally, biofilms were protected from cell death when supplemented with antioxidants and oxidant scavengers, including catalase, mannitol and Tempol. Knocking out SOD (sodAB) in P. aeruginosa led to an enhanced DC effect. Microarray analysis of P. aeruginosa PAO1 showed transcriptional changes in genes related to the stress response and cell death. In conclusion, the electricidal effect results in death of bacteria in biofilms, mediated, at least in part, by production of ROS.

  17. Anhydride-functional silane immobilized onto titanium surfaces induces osteoblast cell differentiation and reduces bacterial adhesion and biofilm formation

    Energy Technology Data Exchange (ETDEWEB)

    Godoy-Gallardo, Maria, E-mail: maria.godoy.gallardo@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Guillem-Marti, Jordi, E-mail: jordi.guillem.marti@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Sevilla, Pablo, E-mail: psevilla@euss.es [Department of Mechanics, Escola Universitària Salesiana de Sarrià (EUSS), C/ Passeig de Sant Bosco, 42, 08017 Barcelona (Spain); Manero, José M., E-mail: jose.maria.manero@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Gil, Francisco J., E-mail: francesc.xavier.gil@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); and others

    2016-02-01

    Bacterial infection in dental implants along with osseointegration failure usually leads to loss of the device. Bioactive molecules with antibacterial properties can be attached to titanium surfaces with anchoring molecules such as silanes, preventing biofilm formation and improving osseointegration. Properties of silanes as molecular binders have been thoroughly studied, but research on the biological effects of these coatings is scarce. The aim of the present study was to determine the in vitro cell response and antibacterial effects of triethoxysilypropyl succinic anhydride (TESPSA) silane anchored on titanium surfaces. X-ray photoelectron spectroscopy confirmed a successful silanization. The silanized surfaces showed no cytotoxic effects. Gene expression analyses of Sarcoma Osteogenic (SaOS-2) osteoblast-like cells cultured on TESPSA silanized surfaces reported a remarkable increase of biochemical markers related to induction of osteoblastic cell differentiation. A manifest decrease of bacterial adhesion and biofilm formation at early stages was observed on treated substrates, while favoring cell adhesion and spreading in bacteria–cell co-cultures. Surfaces treated with TESPSA could enhance a biological sealing on implant surfaces against bacteria colonization of underlying tissues. Furthermore, it can be an effective anchoring platform of biomolecules on titanium surfaces with improved osteoblastic differentiation and antibacterial properties. - Highlights: • TESPSA silane induces osteoblast differentiation. • TESPSA reduces bacterial adhesion and biofilm formation. • TESPSA is a promising anchoring platform of biomolecules onto titanium.

  18. Exposure of Bacterial Biofilms to Electrical Current Leads to Cell Death Mediated in Part by Reactive Oxygen Species.

    Directory of Open Access Journals (Sweden)

    Cassandra L Brinkman

    Full Text Available Bacterial biofilms may form on indwelling medical devices such as prosthetic joints, heart valves and catheters, causing challenging-to-treat infections. We have previously described the 'electricidal effect', in which bacterial biofilms are decreased following exposure to direct electrical current. Herein, we sought to determine if the decreased bacterial quantities are due to detachment of biofilms or cell death and to investigate the role that reactive oxygen species (ROS play in the observed effect. Using confocal and electron microscopy and flow cytometry, we found that direct current (DC leads to cell death and changes in the architecture of biofilms formed by Gram-positive and Gram-negative bacteria. Reactive oxygen species (ROS appear to play a role in DC-associated cell death, as there was an increase in ROS-production by Staphylococcus aureus and Staphylococcus epidermidis biofilms following exposure to DC. An increase in the production of ROS response enzymes catalase and superoxide dismutase (SOD was observed for S. aureus, S. epidermidis and Pseudomonas aeruginosa biofilms following exposure to DC. Additionally, biofilms were protected from cell death when supplemented with antioxidants and oxidant scavengers, including catalase, mannitol and Tempol. Knocking out SOD (sodAB in P. aeruginosa led to an enhanced DC effect. Microarray analysis of P. aeruginosa PAO1 showed transcriptional changes in genes related to the stress response and cell death. In conclusion, the electricidal effect results in death of bacteria in biofilms, mediated, at least in part, by production of ROS.

  19. Anhydride-functional silane immobilized onto titanium surfaces induces osteoblast cell differentiation and reduces bacterial adhesion and biofilm formation

    International Nuclear Information System (INIS)

    Godoy-Gallardo, Maria; Guillem-Marti, Jordi; Sevilla, Pablo; Manero, José M.; Gil, Francisco J.

    2016-01-01

    Bacterial infection in dental implants along with osseointegration failure usually leads to loss of the device. Bioactive molecules with antibacterial properties can be attached to titanium surfaces with anchoring molecules such as silanes, preventing biofilm formation and improving osseointegration. Properties of silanes as molecular binders have been thoroughly studied, but research on the biological effects of these coatings is scarce. The aim of the present study was to determine the in vitro cell response and antibacterial effects of triethoxysilypropyl succinic anhydride (TESPSA) silane anchored on titanium surfaces. X-ray photoelectron spectroscopy confirmed a successful silanization. The silanized surfaces showed no cytotoxic effects. Gene expression analyses of Sarcoma Osteogenic (SaOS-2) osteoblast-like cells cultured on TESPSA silanized surfaces reported a remarkable increase of biochemical markers related to induction of osteoblastic cell differentiation. A manifest decrease of bacterial adhesion and biofilm formation at early stages was observed on treated substrates, while favoring cell adhesion and spreading in bacteria–cell co-cultures. Surfaces treated with TESPSA could enhance a biological sealing on implant surfaces against bacteria colonization of underlying tissues. Furthermore, it can be an effective anchoring platform of biomolecules on titanium surfaces with improved osteoblastic differentiation and antibacterial properties. - Highlights: • TESPSA silane induces osteoblast differentiation. • TESPSA reduces bacterial adhesion and biofilm formation. • TESPSA is a promising anchoring platform of biomolecules onto titanium.

  20. Bacterial CpG-DNA activates dendritic cells in vivo: T helper cell-independent cytotoxic T cell responses to soluble proteins.

    Science.gov (United States)

    Sparwasser, T; Vabulas, R M; Villmow, B; Lipford, G B; Wagner, H

    2000-12-01

    Receptors for conserved molecular patterns associated with microbial pathogens induce synthesis of co-stimulatory molecules and cytokines in immature dendritic cells (DC), as do antigen-reactive CD4 T helper cells via CD40 signaling. Once activated, antigen-presenting DC may activate CD8 T cell responses in a T helper cell-independent fashion. Using immunostimulatory CpG-oligonucleotides (ODN) mimicking bacterial CpG-DNA, we tested whether CpG-DNA bypasses the need for T helper cells in CTL responses towards proteins by directly activating antigen-presenting DC to transit into professional APC. We describe that immature DC in situ constitutively process soluble proteins and generate CD8 T cell determinants yet CD8 T cell responses remain abortive. Induction of primary antigen-specific CD8 cytotoxic T lymphocyte (CTL)-mediated responses becomes initiated in wild-type as well as T helper cell-deficient mice, provided soluble protein and CpG-ODN are draining into the same lymph node. Specifically we show that CpG-ODN trigger antigen-presenting immature DC within the draining lymph node to acutely up-regulate co-stimulatory molecules and produce IL-12. These results provide new insights for generating in vivo efficient CTL responses to soluble proteins which may influence vaccination strategies.

  1. Bacterial Diversity Associated with Anodic Biofilms in Microbial Fuel Cells Fed with Wastewater

    Directory of Open Access Journals (Sweden)

    Alexander Mora Collazos

    2017-01-01

    Full Text Available This study evaluated the bacterial diversity associated with biofilms formed on the anode of microbial fuel cells (MFC, by analyzing the 16S rRNA gene and observations by scanning electron microscopy. Single chambered MFC were constructed and kept in operation for 30 days using environmental samples as inoculum and sole energy substrate; the MFC were monitored as a function of energy production in the course of the experiment; at endpoint, molecular characterization and observations using scanning electron microscopy was performed to the formed biofilms. Values of maximum power density of 4.85 mW/m2 for domestic wastewater and 1.85 mW/m2 in the case of industrial wastewater are reported, with declines of 71 % of the BOD for domestic wastewater and 59 % of the BOD in the case of industrial wastewater. Recovery of 15 unique sequences from the amplification of 16S rRNA gene obtained from the biofilms formed on the anodes was accomplished. Phylogenetic analysis placed these sequences in the Deltaproteobacteria class. The two environmental substrates contain an important and interesting microbial diversity, showing them very promising for the construction and operation of MFC and implementing biodegradation of organic material.

  2. [Effects of UV-induced DNA damage on vector ligation and transformation into bacterial cells].

    Science.gov (United States)

    Huang, Wan-ling; Li, Chang-zheng; Chen, Zhen-rui; He, Wei; Zhou, Ye; Zhou, Zhi-gang; Liu, Shu-wen; Zhou, Chen

    2010-01-01

    To study the effects of UV irradiation on DNA ligation and transformation efficiency of the expression vector into competent bacterial cells. The expression vector was digested with the restriction enzyme SfiI, and the purified target DNA fragments were exposed to UV light at different wavelengths. Ligation and transformation experiments with the exposed fragments were carried out and the colony number and transformation efficiency were assessed. The transformation efficiency of the DNA with a 5-min exposure to 302 nm UV was 60 colonies per nanogram of the DNA, as compared with 20400 for the DNA exposed to 365 nm UV. The time course experiment showed that prolonged DNA exposure to 365 nm UV light was associated with lowered transformation efficiency. DNA exposure for 30 min caused a reduction of the transformation efficiency to lower than 50% compared to that of DNA without UV exposure. But with a 15 min exposure, the DNA maintained a transformation efficiency more than 70%, which was sufficient for most molecular biology experiments. In construction of the expression vector, it is advisable to prevent the target DNA from UV exposure. When UV exposure is essential, we suggest that 365 nm UV be used and the exposure time controlled within 15 min.

  3. Effects of blood processing and sample storage on the stability of biotherapeutics and anti-drug antibodies.

    Science.gov (United States)

    Andisik, Matthew; DeStefano, Lisa; Stefan, Colin; Gathani, Meghna; Laurino, Kevin; Lai, Vicky Ch; Rajadhyaksha, Manoj; Torri, Albert; Shao, Weiping

    2015-01-01

    Pre-analytical factors such as sample processing, handling or storage could affect the stability of biotherapeutics and anti-drug antibodies in clinical samples, potentially impacting the pharmacokinetic and immunogenicity assessments. We used sarilumab, a fully human IgG1 monoclonal antibody, and evaluated the stability of sarilumab (both functional and bound forms) and anti-sarilumab antibodies in blood samples during serum collection and the impact of various processing conditions on the analyte stability in serum for long-term storage. We also assessed the incurred sample stability of these analytes in samples from clinical studies. Assessment of analyte stability can provide relevant information about sample stability under different pre-analytical conditions and improve the confidence in the validity of bioanalytical data generated.

  4. Heterotrophic free-living and particle-bound bacterial cell size in the ...

    Indian Academy of Sciences (India)

    PRAKASH

    Aquatic Microbial Ecology Research Laboratory, Department of Studies in Zoology, University of Mysore,. Manasagangotri, Mysore 570 ... Regression analysis revealed that 18% of the variation in mean heterotrophic free-living bacterial .... multiple regression analysis was also used with bacterial variables as dependent ...

  5. Targeted liposomes for cytosolic drug delivery to tumor cells

    NARCIS (Netherlands)

    Mastrobattista, E.

    2001-01-01

    In this thesis, a Trojan horse strategy with antibody-targeted liposomes has been followed to obtain cytosolic delivery of biotherapeutics to tumor cells in vitro. This strategy involves targeting of immunoliposomes to specific receptors on tumor cells that result in receptor-mediated uptake of the

  6. Wholly Rickettsia! Reconstructed Metabolic Profile of the Quintessential Bacterial Parasite of Eukaryotic Cells

    Directory of Open Access Journals (Sweden)

    Timothy P. Driscoll

    2017-09-01

    Full Text Available Reductive genome evolution has purged many metabolic pathways from obligate intracellular Rickettsia (Alphaproteobacteria; Rickettsiaceae. While some aspects of host-dependent rickettsial metabolism have been characterized, the array of host-acquired metabolites and their cognate transporters remains unknown. This dearth of information has thwarted efforts to obtain an axenic Rickettsia culture, a major impediment to conventional genetic approaches. Using phylogenomics and computational pathway analysis, we reconstructed the Rickettsia metabolic and transport network, identifying 51 host-acquired metabolites (only 21 previously characterized needed to compensate for degraded biosynthesis pathways. In the absence of glycolysis and the pentose phosphate pathway, cell envelope glycoconjugates are synthesized from three imported host sugars, with a range of additional host-acquired metabolites fueling the tricarboxylic acid cycle. Fatty acid and glycerophospholipid pathways also initiate from host precursors, and import of both isoprenes and terpenoids is required for the synthesis of ubiquinone and the lipid carrier of lipid I and O-antigen. Unlike metabolite-provisioning bacterial symbionts of arthropods, rickettsiae cannot synthesize B vitamins or most other cofactors, accentuating their parasitic nature. Six biosynthesis pathways contain holes (missing enzymes; similar patterns in taxonomically diverse bacteria suggest alternative enzymes that await discovery. A paucity of characterized and predicted transporters emphasizes the knowledge gap concerning how rickettsiae import host metabolites, some of which are large and not known to be transported by bacteria. Collectively, our reconstructed metabolic network offers clues to how rickettsiae hijack host metabolic pathways. This blueprint for growth determinants is an important step toward the design of axenic media to rescue rickettsiae from the eukaryotic cell.

  7. Hierarchical architecture of bacterial cellulose and composite plant cell wall polysaccharide hydrogels using small angle neutron scattering.

    Science.gov (United States)

    Martínez-Sanz, Marta; Gidley, Michael J; Gilbert, Elliot P

    2016-02-07

    Small angle neutron scattering (SANS) has been applied to characterise the structure of pure bacterial cellulose hydrogels, and composites thereof, with two plant cell wall polysaccharides (arabinoxylan and xyloglucan). Conventional published models, which assume that bacterial cellulose ribbons are solid one-phase systems, fail to adequately describe the SANS data of pure bacterial cellulose. Fitting of the neutron scattering profiles instead suggests that the sub-structure of cellulose microfibrils contained within the ribbons results in the creation of regions with distinct values of neutron scattering length density, when the hydrogels are subjected to H2O/D2O exchange. This may be represented within a core-shell formalism that considers the cellulose ribbons to comprise a core containing impermeable crystallites surrounded by a network of paracrystalline cellulose and tightly bound water, and a shell containing only paracrystalline cellulose and water. Accordingly, a fitting function comprising the sum of a power-law term to account for the large scale structure of intertwined ribbons, plus a core-shell cylinder with polydisperse radius, has been applied; it is demonstrated to simultaneously describe all SANS contrast variation data of pure and composite bacterial cellulose hydrogels. In addition, the resultant fitting parameters indicate distinct interaction mechanisms of arabinoxylan and xyloglucan with cellulose, revealing the potential of this approach to investigate the role of different plant cell wall polysaccharides on the biosynthesis process of cellulose.

  8. Fundamental principles in bacterial physiology—history, recent progress, and the future with focus on cell size control: a review

    Science.gov (United States)

    Jun, Suckjoon; Si, Fangwei; Pugatch, Rami; Scott, Matthew

    2018-05-01

    Bacterial physiology is a branch of biology that aims to understand overarching principles of cellular reproduction. Many important issues in bacterial physiology are inherently quantitative, and major contributors to the field have often brought together tools and ways of thinking from multiple disciplines. This article presents a comprehensive overview of major ideas and approaches developed since the early 20th century for anyone who is interested in the fundamental problems in bacterial physiology. This article is divided into two parts. In the first part (sections 1–3), we review the first ‘golden era’ of bacterial physiology from the 1940s to early 1970s and provide a complete list of major references from that period. In the second part (sections 4–7), we explain how the pioneering work from the first golden era has influenced various rediscoveries of general quantitative principles and significant further development in modern bacterial physiology. Specifically, section 4 presents the history and current progress of the ‘adder’ principle of cell size homeostasis. Section 5 discusses the implications of coarse-graining the cellular protein composition, and how the coarse-grained proteome ‘sectors’ re-balance under different growth conditions. Section 6 focuses on physiological invariants, and explains how they are the key to understanding the coordination between growth and the cell cycle underlying cell size control in steady-state growth. Section 7 overviews how the temporal organization of all the internal processes enables balanced growth. In the final section 8, we conclude by discussing the remaining challenges for the future in the field.

  9. Intravitreal concentrations of a near-infrared fluorescence-labeled biotherapeutic determined in situ using confocal scanning laser ophthalmoscopy.

    Science.gov (United States)

    Basile, Anthony S; Glazier, Genevieve; Lee, Alice; Jiang, Li-Ying; Johnson, Theodore R; Shields, Michael J; Vezina, Mark; Doppalapudi, Venkata R

    2011-09-01

    The pharmacokinetics of ophthalmic biotherapeutics are difficult to determine in human vitreous humor. Because of the high transparency of living tissue to near-infrared (NIR) light, the temporal changes in vitreous concentrations of a biomolecule labeled with an NIR fluorescent probe can be monitored in situ with a scanning laser ophthalmoscope (SLO). A humanized IgG was labeled with the NIR probe IRDye800CW (CVX-4164). Rabbits were given CVX-4164 intravitreally, and NIR fluorescence intensity was measured in the central plane of the vitreous humor with an SLO. Fluorescence intensities were converted to concentrations by using standard curves. Little background fluorescence was detected, and the minimum detectable concentration of CVX-4164 was CVX-4164 determined in situ declined with time, with C(max) ≈ 1 μM and t½ = 145 hours (112-μg dose). The t½ of CVX-4164 was approximately three times greater than that of the IRDye800CW alone, whereas the vitreal clearance (CL) and volume of distribution (V(ss)) of the native dye were approximately 2000- and 550-fold greater than that of the conjugate. CVX-4164 concentrations determined in situ were 2.6 to 4.4 times higher than those determined by ex vivo NIR fluorescence or ELISA in homogenized vitreous humor, reflecting the greater spatial resolution of in situ imaging. Moreover, vitreal concentrations determined in situ were >3 orders of magnitude greater than plasma concentrations of CVX-4164, as determined by ELISA, and had a different kinetic profile. This study demonstrates the feasibility of determining the pharmacokinetics of intraocular biotherapeutics labeled with NIR fluorescent probes by in situ monitoring.

  10. Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

    International Nuclear Information System (INIS)

    Fiedler, Tomas; Salamon, Achim; Adam, Stefanie; Herzmann, Nicole; Taubenheim, Jan; Peters, Kirsten

    2013-01-01

    Adult mesenchymal stem cells (MSC) are present in several tissues, e.g. bone marrow, heart muscle, brain and subcutaneous adipose tissue. In invasive infections MSC get in contact with bacteria and bacterial components. Not much is known about how bacterial pathogens interact with MSC and how contact to bacteria influences MSC viability and differentiation potential. In this study we investigated the impact of three different wound infection relevant bacteria, Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes, and the cell wall components lipopolysaccharide (LPS; Gram-negative bacteria) and lipoteichoic acid (LTA; Gram-positive bacteria) on viability, proliferation, and osteogenic as well as adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (adMSC). We show that all three tested species were able to attach to and internalize into adMSC. The heat-inactivated Gram-negative E. coli as well as LPS were able to induce proliferation and osteogenic differentiation but reduce adipogenic differentiation of adMSC. Conspicuously, the heat-inactivated Gram-positive species showed the same effects on proliferation and adipogenic differentiation, while its cell wall component LTA exhibited no significant impact on adMSC. Therefore, our data demonstrate that osteogenic and adipogenic differentiation of adMSC is influenced in an oppositional fashion by bacterial antigens and that MSC-governed regeneration is not necessarily reduced under infectious conditions. - Highlights: • Staphylococcus aureus, Streptococcus pyogenes and Escherichia coli bind to and internalize into adMSC. • Heat-inactivated cells of these bacterial species trigger proliferation of adMSC. • Heat-inactivated E. coli and LPS induce osteogenic differentiation of adMSC. • Heat-inactivated E. coli and LPS reduce adipogenic differentiation of adMSC. • LTA does not influence adipogenic or osteogenic differentiation of adMSC

  11. Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Fiedler, Tomas, E-mail: tomas.fiedler@med.uni-rostock.de [Institute for Medical Microbiology, Virology, and Hygiene, Rostock University Medical Center, Schillingallee 70, D-18057 Rostock (Germany); Salamon, Achim; Adam, Stefanie; Herzmann, Nicole [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Taubenheim, Jan [Institute for Medical Microbiology, Virology, and Hygiene, Rostock University Medical Center, Schillingallee 70, D-18057 Rostock (Germany); Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Peters, Kirsten [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany)

    2013-11-01

    Adult mesenchymal stem cells (MSC) are present in several tissues, e.g. bone marrow, heart muscle, brain and subcutaneous adipose tissue. In invasive infections MSC get in contact with bacteria and bacterial components. Not much is known about how bacterial pathogens interact with MSC and how contact to bacteria influences MSC viability and differentiation potential. In this study we investigated the impact of three different wound infection relevant bacteria, Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes, and the cell wall components lipopolysaccharide (LPS; Gram-negative bacteria) and lipoteichoic acid (LTA; Gram-positive bacteria) on viability, proliferation, and osteogenic as well as adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (adMSC). We show that all three tested species were able to attach to and internalize into adMSC. The heat-inactivated Gram-negative E. coli as well as LPS were able to induce proliferation and osteogenic differentiation but reduce adipogenic differentiation of adMSC. Conspicuously, the heat-inactivated Gram-positive species showed the same effects on proliferation and adipogenic differentiation, while its cell wall component LTA exhibited no significant impact on adMSC. Therefore, our data demonstrate that osteogenic and adipogenic differentiation of adMSC is influenced in an oppositional fashion by bacterial antigens and that MSC-governed regeneration is not necessarily reduced under infectious conditions. - Highlights: • Staphylococcus aureus, Streptococcus pyogenes and Escherichia coli bind to and internalize into adMSC. • Heat-inactivated cells of these bacterial species trigger proliferation of adMSC. • Heat-inactivated E. coli and LPS induce osteogenic differentiation of adMSC. • Heat-inactivated E. coli and LPS reduce adipogenic differentiation of adMSC. • LTA does not influence adipogenic or osteogenic differentiation of adMSC.

  12. [Preoperatiove Airway Bacterial Colonization: the Missing Link between Non-small Cell Lung Cancer Following Lobectomy and Postoperative Pneumonia?

    Science.gov (United States)

    Gao, Ke; Lai, Yutian; Huang, Jian; Wang, Yifan; Wang, Xiaowei; Che, Guowei

    2017-04-20

    Surgical procedure is the main method of treating lung cancer. Meanwhile, postoperative pneumonia (POP) is the major cause of perioperative mortality in lung cancer surgery. The preoperative pathogenic airway bacterial colonization is an independent risk factor causing postoperative pulmonary complications (PPC). This cross-sectional study aimed to explore the relationship between preoperative pathogenic airway bacterial colonization and POP in lung cancer and to identify the high-risk factors of preoperative pathogenic airway bacterial colonization. A total of 125 patients with non-small cell lung cancer (NSCLC) underwent thoracic surgery in six hospitals of Chengdu between May 2015 and January 2016. Preoperative pathogenic airway bacterial colonization was detected in all patients via fiber bronchoscopy. Patients' PPC, high-risk factors, clinical characteristics, and the serum surfactant protein D (SP-D) level were also analyzed. The incidence of preoperative pathogenic airway bacterial colonization among NSCLC patients was 15.2% (19/125). Up to 22 strains were identified in the colonization positive group, with Gram-negative bacteria being dominant (86.36%, 19/22). High-risk factors of pathogenic airway bacterial colonization were age (≥75 yr) and smoking index (≥400 cigarettes/year). PPC incidence was significantly higher in the colonization-positive group (42.11%, 8/19) than that in the colonization-negative group (16.04%, 17/106)(P=0.021). POP incidence was significantly higher in the colonization-positive group (26.32%, 5/19) than that in the colonization-negative group (6.60%, 7/106)(P=0.019). The serum SP-D level of patients in the colonization-positive group was remarkably higher than that in the colonization-negative group [(31.25±6.09) vs (28.17±5.23)](P=0.023). The incidence of preoperative pathogenic airway bacterial colonization among NSCLC patients with POP was 41.67% (5/12). This value was 3.4 times higher than that among the patients without

  13. Bacterial Adhesion & Blocking Bacterial Adhesion

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk

    2008-01-01

    reduce or delay bacterial biofilm formation of a range of urinary tract infectious E.coli and Klebsiella isolates. Several other proteinaceous coatings were also found to display anti-adhesive properties, possibly providing a measure for controlling the colonization of implant materials. Several other...... components. These substances may both mediate and stabilize the bacterial biofilm. Finally, several adhesive structures were examined, and a novel physiological biofilm phenotype in E.coli biofilms was characterized, namely cell chain formation. The autotransporter protein, antigen 43, was implicated...

  14. Natural Killer Cell-Mediated Host Defense against Uropathogenic E. coli Is Counteracted by Bacterial HemolysinA-Dependent Killing of NK Cells

    Science.gov (United States)

    Gur, Chamutal; Coppenhagen-Glazer, Shunit; Rosenberg, Shilo; Yamin, Rachel; Enk, Jonatan; Glasner, Ariella; Bar-On, Yotam; Fleissig, Omer; Naor, Ronit; Abed, Jawad; Mevorach, Dror; Granot, Zvi; Bachrach, Gilad; Mandelboim, Ofer

    2013-01-01

    SUMMARY Uropathogenic Escherichia coli (UPEC) are a common cause of urinary tract infections (UTIs) in humans. While the importance of natural killer (NK) cells in innate immune protection against tumors and viral infections is well documented, their role in defense against bacterial infections is still emerging, and their involvement in UPEC-mediated UTI is practically unknown. Using a systematic mutagenesis approach, we found that UPEC adheres to NK cells primarily via its type I fimbriae and employs its hemolysinA toxin to kill NK cells. In the absence of hemolysinA, NK cells directly respond to the bacteria and secrete the cytokine TNF-α, which results in decreased bacterial numbers in vitro and reduction of bacterial burden in the infected bladders. Thus, NK cells control UPEC via TNF-α production, which UPEC counteracts by hemolysinA-mediated killing of NK cells, representing a previously unrecognized host defense and microbial counterattack mechanism in the context of UTI. PMID:24331464

  15. Phage-Bacterial Dynamics with Spatial Structure: Self Organization around Phage Sinks Can Promote Increased Cell Densities.

    Science.gov (United States)

    Bull, James J; Christensen, Kelly A; Scott, Carly; Jack, Benjamin R; Crandall, Cameron J; Krone, Stephen M

    2018-01-29

    Bacteria growing on surfaces appear to be profoundly more resistant to control by lytic bacteriophages than do the same cells grown in liquid. Here, we use simulation models to investigate whether spatial structure per se can account for this increased cell density in the presence of phages. A measure is derived for comparing cell densities between growth in spatially structured environments versus well mixed environments (known as mass action). Maintenance of sensitive cells requires some form of phage death; we invoke death mechanisms that are spatially fixed, as if produced by cells. Spatially structured phage death provides cells with a means of protection that can boost cell densities an order of magnitude above that attained under mass action, although the effect is sometimes in the opposite direction. Phage and bacteria self organize into separate refuges, and spatial structure operates so that the phage progeny from a single burst do not have independent fates (as they do with mass action). Phage incur a high loss when invading protected areas that have high cell densities, resulting in greater protection for the cells. By the same metric, mass action dynamics either show no sustained bacterial elevation or oscillate between states of low and high cell densities and an elevated average. The elevated cell densities observed in models with spatial structure do not approach the empirically observed increased density of cells in structured environments with phages (which can be many orders of magnitude), so the empirical phenomenon likely requires additional mechanisms than those analyzed here.

  16. Bacterial cell-surface displaying of thermo-tolerant glutamate dehydrogenase and its application in L-glutamate assay.

    Science.gov (United States)

    Song, Jianxia; Liang, Bo; Han, Dongfei; Tang, Xiangjiang; Lang, Qiaolin; Feng, Ruirui; Han, Lihui; Liu, Aihua

    2015-03-01

    In this paper, glutamate dehydrogenase (Gldh) is reported to efficiently display on Escherichia coli cell surface by using N-terminal region of ice the nucleation protein as an anchoring motif. The presence of Gldh was confirmed by SDS-PAGE and enzyme activity assay. Gldh was detected mainly in the outer membrane fraction, suggesting that the Gldh was displayed on the bacterial cell surface. The optimal temperature and pH for the bacteria cell-surface displayed Gldh (bacteria-Gldh) were 70°C and 9.0, respectively. Additionally, the fusion protein retained almost 100% of its initial enzymatic activity after 1 month incubation at 4°C. Transition metal ions could inhibit the enzyme activity to different extents, while common anions had little adverse effect on enzyme activity. Importantly, the displayed Gldh is most specific to l-glutamate reported so far. The bacterial Gldh was enabled to catalyze oxidization of l-glutamate with NADP(+) as cofactor, and the resultant NADPH can be detected spectrometrically at 340nm. The bacterial-Gldh based l-glutamate assay was established, where the absorbance at 340nm increased linearly with the increasing l-glutamate concentration within the range of 10-400μM. Further, the proposed approach was successfully applied to measure l-glutamate in real samples. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Identification and monitoring of host cell proteins by mass spectrometry combined with high performance immunochemistry testing.

    Directory of Open Access Journals (Sweden)

    Katrin Bomans

    Full Text Available Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS. However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day manner.

  18. Bioelectrogenesis with microbial fuel cells (MFCs using the microalga Chlorella vulgaris and bacterial communities

    Directory of Open Access Journals (Sweden)

    Ronald Huarachi-Olivera

    2018-01-01

    Conclusions: These findings suggest that MFCs with C. vulgaris and bacterial community have a simultaneous efficiency in the production of bioelectricity and bioremediation processes, becoming an important source of bioenergy in the future.

  19. Biotherapeutic protein formulation variables influence protein integrity and can promote post-translational modifications as shown using chicken egg white lysozyme as a model system.

    Science.gov (United States)

    Gourbatsi, Evdoxia; Povey, Jane; Uddin, Shahid; Smales, C Mark

    2016-04-01

    The effect of different formulations variables on protein integrity were investigated using lysozyme as a model protein for the development of biotherapeutic protein formulations for use in the clinic. Buffer composition/concentration was the key variable of formulation reagents investigated in determining lysozyme stability and authenticity independent of protein concentration whilst the storage temperature and time, not surprisingly, were also key variables. Tryptic peptide mapping of the protein showed that the modifications occurred when formulated under specific conditions but not others. A model peptide system was developed that reflected the same behavior under formulation conditions as intact lysozyme. Peptide models may mirror the stability of proteins, or regions of proteins, in the same formulations and be used to help develop a rapid screen of formulations for stabilisation of biotherapeutic proteins.

  20. Yeast cell wall extract induces disease resistance against bacterial and fungal pathogens in Arabidopsis thaliana and Brassica crop.

    Directory of Open Access Journals (Sweden)

    Mari Narusaka

    Full Text Available Housaku Monogatari (HM is a plant activator prepared from a yeast cell wall extract. We examined the efficacy of HM application and observed that HM treatment increased the resistance of Arabidopsis thaliana and Brassica rapa leaves to bacterial and fungal infections. HM reduced the severity of bacterial leaf spot and anthracnose on A. thaliana and Brassica crop leaves with protective effects. In addition, gene expression analysis of A. thaliana plants after treatment with HM indicated increased expression of several plant defense-related genes. HM treatment appears to induce early activation of jasmonate/ethylene and late activation of salicylic acid (SA pathways. Analysis using signaling mutants revealed that HM required SA accumulation and SA signaling to facilitate resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola and the fungal pathogen Colletotrichum higginsianum. In addition, HM-induced resistance conferred chitin-independent disease resistance to bacterial pathogens in A. thaliana. These results suggest that HM contains multiple microbe-associated molecular patterns that activate defense responses in plants. These findings suggest that the application of HM is a useful tool that may facilitate new disease control methods.

  1. Biotherapeutic protein formulation variables influence protein integrity and can promote post-translational modifications as shown using chicken egg white lysozyme as a model system

    OpenAIRE

    Gourbatsi, Evdoxia; Povey, Jane; Uddin, Shahid; Smales, C. Mark

    2015-01-01

    Objectives The effect of different formulations variables on protein integrity were investigated using lysozyme as a model protein for the development of biotherapeutic protein formulations for use in the clinic. Results Buffer composition/concentration was the key variable of formulation reagents investigated in determining lysozyme stability and authenticity independent of protein concentration whilst the storage temperature and time, not surprisingly, were also key variables. Tryptic pepti...

  2. Bacterial Growth State Distinguished by Single-Cell Protein Profiling: Does Chlorination Kill Coliforms in Municipal Effluent?

    Science.gov (United States)

    Rockabrand, David; Austin, Teresa; Kaiser, Robyn; Blum, Paul

    1999-01-01

    Municipal effluent is the largest reservoir of human enteric bacteria. Its public health significance, however, depends upon the physiological status of the wastewater bacterial community. A novel immunofluorescence assay was developed and used to examine the bacterial growth state during wastewater disinfection. Quantitative levels of three highly conserved cytosolic proteins (DnaK, Dps, and Fis) were determined by using enterobacterium-specific antibody fluorochrome-coupled probes. Enterobacterial Fis homologs were abundant in growing cells and nearly undetectable in stationary-phase cells. In contrast, enterobacterial Dps homologs were abundant in stationary-phase cells but virtually undetectable in growing cells. The range of variation in the abundance of both proteins was at least 100-fold as determined by Western blotting and immunofluorescence analysis. Enterobacterial DnaK homologs were nearly invariant with growth state, enabling their use as permeabilization controls. The cellular growth states of individual enterobacteria in wastewater samples were determined by measurement of Fis, Dps, and DnaK abundance (protein profiling). Intermediate levels of Fis and Dps were evident and occurred in response to physiological transitions. The results indicate that chlorination failed to kill coliforms but rather elicited nutrient starvation and a reversible nonculturable state. These studies suggest that the current standard procedures for wastewater analysis which rely on detection of culturable cells likely underestimate fecal coliform content. PMID:10473432

  3. Sonic Hedgehog Signaling Regulates Hematopoietic Stem/Progenitor Cell Activation during the Granulopoietic Response to Systemic Bacterial Infection.

    Science.gov (United States)

    Shi, Xin; Wei, Shengcai; Simms, Kevin J; Cumpston, Devan N; Ewing, Thomas J; Zhang, Ping

    2018-01-01

    Activation and reprogramming of hematopoietic stem/progenitor cells play a critical role in the granulopoietic response to bacterial infection. Our current study determined the significance of Sonic hedgehog (SHH) signaling in the regulation of hematopoietic precursor cell activity during the host defense response to systemic bacterial infection. Bacteremia was induced in male Balb/c mice via intravenous injection (i.v.) of Escherichia coli (5 × 10 7 CFUs/mouse). Control mice received i.v. saline. SHH protein level in bone marrow cell (BMC) lysates was markedly increased at both 24 and 48 h of bacteremia. By contrast, the amount of soluble SHH ligand in marrow elutes was significantly reduced. These contrasting alterations suggested that SHH ligand release from BMCs was reduced and/or binding of soluble SHH ligand to BMCs was enhanced. At both 12 and 24 h of bacteremia, SHH mRNA expression by BMCs was significantly upregulated. This upregulation of SHH mRNA expression was followed by a marked increase in SHH protein expression in BMCs. Activation of the ERK1/2-SP1 pathway was involved in mediating the upregulation of SHH gene expression. The major cell type showing the enhancement of SHH expression in the bone marrow was lineage positive cells. Gli1 positioned downstream of the SHH receptor activation serves as a key component of the hedgehog (HH) pathway. Primitive hematopoietic precursor cells exhibited the highest level of baseline Gli1 expression, suggesting that they were active cells responding to SHH ligand stimulation. Along with the increased expression of SHH in the bone marrow, expression of Gli1 by marrow cells was significantly upregulated at both mRNA and protein levels following bacteremia. This enhancement of Gli1 expression was correlated with activation of hematopoietic stem/progenitor cell proliferation. Mice with Gli1 gene deletion showed attenuation in activation of marrow hematopoietic stem/progenitor cell proliferation and inhibition

  4. Effect of Bacterial Lipopolysaccharide Contamination on Gutta Percha- versus Resilon-Induced Human Monocyte Cell Line Toxicity.

    Directory of Open Access Journals (Sweden)

    Jamshid Hadjati

    2015-04-01

    Full Text Available Cytotoxic effects of obturation materials were tested in presence and absence of endotoxin on human monocytes in vitro.Human monocytes from THP-1 cell line were cultured. Three millimeters from the tip of each Resilon and gutta percha points were cut and directly placed at the bottom of the culture wells. Cultured cells were exposed to gutta percha (groups G1 and G2 and Resilon (R1 and R2. Ten μg/ml bacterial lipopolysaccharide (LPS was added to the culture wells in groups G1 and R1. Positive control included the bacterial LPS without the root canal filling material and the negative control contained the cells in culture medium only. Viability of cells was tested in all groups after 24, 48, and 72 hours using the methylthiazolyldiphenyl-tetrazolium bromide (MTT assay for at least 3 times to obtain reproducible results. Optical density values were read and the data were analyzed using three-way ANOVA and post hoc statistical test.The results showed that cells in G2 had the lowest rate of viability at 24 hours, but the lowest rate of viable cells was recorded in G1 at 48 and 72 hours. The effect of LPS treatment was not statistically significant. Resilon groups showed cell viability values higher than those of gutta percha groups, although statistically non-significant (P=0.105. Cell viability values were lower in gutta percha than Resilon groups when LPS-treated and LPS-untreated groups were compared independently at each time point.It could be concluded that none of the tested root canal filling materials had toxic effects on cultured human monocyte cells whether in presence or absence of LPS contamination.

  5. New Parameters to Quantitatively Express the Invasiveness of Bacterial Strains from Implant-Related Orthopaedic Infections into Osteoblast Cells

    Directory of Open Access Journals (Sweden)

    Davide Campoccia

    2018-04-01

    Full Text Available Complete eradication of bacterial infections is often a challenging task, especially in presence of prosthetic devices. Invasion of non-phagocytic host cells appears to be a critical mechanism of microbial persistence in host tissues. Hidden within host cells, bacteria elude host defences and antibiotic treatments that are intracellularly inactive. The intracellular invasiveness of bacteria is generally measured by conventional gentamicin protection assays. The efficiency of invasion, however, markedly differs across bacterial species and adjustments to the titre of the microbial inocula used in the assays are often needed to enumerate intracellular bacteria. Such changes affect the standardisation of the method and hamper a direct comparison of bacteria on a same scale. This study aims at investigating the precise relation between inoculum, in terms of multiplicity of infection (MOI, and internalised bacteria. The investigation included nine Staphylococcus aureus, seven Staphylococcus epidermidis, five Staphylococcus lugdunensis and two Enterococcus faecalis clinical strains, which are co-cultured with MG63 human osteoblasts. Unprecedented insights are offered on the relations existing between MOI, number of internalised bacteria and per cent of internalised bacteria. New parameters are identified that are of potential use for qualifying the efficiency of internalization and compare the behaviour of bacterial strains.

  6. New Parameters to Quantitatively Express the Invasiveness of Bacterial Strains from Implant-Related Orthopaedic Infections into Osteoblast Cells.

    Science.gov (United States)

    Campoccia, Davide; Montanaro, Lucio; Ravaioli, Stefano; Cangini, Ilaria; Testoni, Francesca; Visai, Livia; Arciola, Carla Renata

    2018-04-03

    Complete eradication of bacterial infections is often a challenging task, especially in presence of prosthetic devices. Invasion of non-phagocytic host cells appears to be a critical mechanism of microbial persistence in host tissues. Hidden within host cells, bacteria elude host defences and antibiotic treatments that are intracellularly inactive. The intracellular invasiveness of bacteria is generally measured by conventional gentamicin protection assays. The efficiency of invasion, however, markedly differs across bacterial species and adjustments to the titre of the microbial inocula used in the assays are often needed to enumerate intracellular bacteria. Such changes affect the standardisation of the method and hamper a direct comparison of bacteria on a same scale. This study aims at investigating the precise relation between inoculum, in terms of multiplicity of infection (MOI), and internalised bacteria. The investigation included nine Staphylococcus aureus , seven Staphylococcus epidermidis , five Staphylococcus lugdunensis and two Enterococcus faecalis clinical strains, which are co-cultured with MG63 human osteoblasts. Unprecedented insights are offered on the relations existing between MOI, number of internalised bacteria and per cent of internalised bacteria. New parameters are identified that are of potential use for qualifying the efficiency of internalization and compare the behaviour of bacterial strains.

  7. One size does not fit all--bacterial cell death by antibiotics cannot be explained by the action of reactive oxygen species.

    Science.gov (United States)

    Kuhnert, Nikolai

    2013-10-11

    Back to square one: Two recent studies prove that reactive oxygen species (ROS) are not responsible for bacterial cell death after treatment with antibiotics. The ROS concept cannot be generalized to explain all processes resulting in cell death. The search for the mechanism of action of bacterial antibiotics must thus return to the beginning. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Dissecting Bacterial Cell Wall Entry and Signaling in Eukaryotic Cells: an Actin-Dependent Pathway Parallels Platelet-Activating Factor Receptor-Mediated Endocytosis.

    Science.gov (United States)

    Loh, Lip Nam; Gao, Geli; Tuomanen, Elaine I

    2017-01-03

    The Gram-positive bacterial cell wall (CW) peptidoglycan-teichoic acid complex is released into the host environment during bacterial metabolism or death. It is a highly inflammatory Toll-like receptor 2 (TLR2) ligand, and previous in vivo studies have demonstrated its ability to recapitulate pathological features of pneumonia and meningitis. We report that an actin-dependent pathway is involved in the internalization of the CW by epithelial and endothelial cells, in addition to the previously described platelet-activating factor receptor (PAFr)-dependent uptake pathway. Unlike the PAFr-dependent pathway, which is mediated by clathrin and dynamin and does not lead to signaling, the alternative pathway is sensitive to 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and engenders Rac1, Cdc42, and phosphatidylinositol 3-kinase (PI3K) signaling. Upon internalization by this macropinocytosis-like pathway, CW is trafficked to lysosomes. Intracellular CW trafficking is more complex than previously recognized and suggests multiple points of interaction with and without innate immune signaling. Streptococcus pneumoniae is a major human pathogen infecting the respiratory tract and brain. It is an established model organism for understanding how infection injures the host. During infection or bacterial growth, bacteria shed their cell wall (CW) into the host environment and trigger inflammation. A previous study has shown that CW enters and crosses cell barriers by interacting with a receptor on the surfaces of host cells, termed platelet-activating factor receptor (PAFr). In the present study, by using cells that are depleted of PAFr, we identified a second pathway with features of macropinocytosis, which is a receptor-independent fluid uptake mechanism by cells. Each pathway contributes approximately the same amount of cell wall trafficking, but the PAFr pathway is silent, while the new pathway appears to contribute to the host inflammatory response to CW insult. Copyright © 2017

  9. Bacterial Cell Wall Precursor Phosphatase Assays Using Thin-layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC).

    Science.gov (United States)

    Pazos, Manuel; Otten, Christian; Vollmer, Waldemar

    2018-03-20

    Peptidoglycan encases the bacterial cytoplasmic membrane to protect the cell from lysis due to the turgor. The final steps of peptidoglycan synthesis require a membrane-anchored substrate called lipid II, in which the peptidoglycan subunit is linked to the carrier lipid undecaprenol via a pyrophosphate moiety. Lipid II is the target of glycopeptide antibiotics and several antimicrobial peptides, and is degraded by 'attacking' enzymes involved in bacterial competition to induce lysis. Here we describe two protocols using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC), respectively, to assay the digestion of lipid II by phosphatases such as Colicin M or the LXG toxin protein TelC from Streptococcus intermedius . The TLC method can also monitor the digestion of undecaprenyl (pyro)phosphate, whereas the HPLC method allows to separate the di-, mono- or unphosphorylated disaccharide pentapeptide products of lipid II.

  10. Transmission electron microscopic analysis showing structural changes to bacterial cells treated with electrolyzed water and an acidic sanitizer.

    Science.gov (United States)

    Feliciano, Lizanel; Lee, Jaesung; Pascall, Melvin A

    2012-04-01

    The effects of various sanitizers on the viability and cellular injury to structures of Escherichia coli and Listeria innocua were investigated. A food grade organic acidic formulation (pH 2.5) and acidic, neutral, and basic electrolyzed water [AEW (pH 2.7, oxidation reduction potential; ORP: 1100 mV, free available chlorine; FAC: 150 ppm), NEW (pH 6.9, ORP: 840 mV, FAC: 150 ppm), BEW (pH 11.6, ORP: -810 mV)] were used to treat E. coli and L. innocua cells. After 10 min of exposure to the sanitizers, changes to the bacterial numbers and cell structures were evaluated by plate counting and transmission electron microscopy (TEM), respectively. It was concluded from the results that the sanitizers reduced the E. coli cells between 2 and 3 log CFU/mL. Except for the BEW treatment, reductions in L. innocua population were greater (>1 log CFU/mL) than that of E. coli for all treatments. Data from the TEM showed that all sanitizers caused changes to the cell envelope and cytoplasm of both organisms. However, smaller changes were observed for L. innocua cells. Decrease in the integrity of the cell envelope and aggregation of the cytoplasmic components appeared to be mainly because of exposure to the sanitizers. The organic acid formulation and AEW were the most effective sanitizers against bacterial cells, indicating that penetration of acidic substances effectively caused the cell inactivation. An understanding of the method in which E-water and an acidic sanitizer cause injury to E. coli and L. innocua would be helpful in selecting an effective chemical agent as a food safety tool. This will allow a scientist to target similar microorganisms such as food borne bacteria with structures that are vulnerable to the sanitizer. © 2012 Institute of Food Technologists®

  11. Crystal structure of bacterial cell-surface alginate-binding protein with an M75 peptidase motif.

    Science.gov (United States)

    Maruyama, Yukie; Ochiai, Akihito; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

    2011-02-18

    A gram-negative Sphingomonas sp. A1 directly incorporates alginate polysaccharide into the cytoplasm via the cell-surface pit and ABC transporter. A cell-surface alginate-binding protein, Algp7, functions as a concentrator of the polysaccharide in the pit. Based on the primary structure and genetic organization in the bacterial genome, Algp7 was found to be homologous to an M75 peptidase motif-containing EfeO, a component of a ferrous ion transporter. Despite the presence of an M75 peptidase motif with high similarity, the Algp7 protein purified from recombinant Escherichia coli cells was inert on insulin B chain and N-benzoyl-Phe-Val-Arg-p-nitroanilide, both of which are substrates for a typical M75 peptidase, imelysin, from Pseudomonas aeruginosa. The X-ray crystallographic structure of Algp7 was determined at 2.10Å resolution by single-wavelength anomalous diffraction. Although a metal-binding motif, HxxE, conserved in zinc ion-dependent M75 peptidases is also found in Algp7, the crystal structure of Algp7 contains no metal even at the motif. The protein consists of two structurally similar up-and-down helical bundles as the basic scaffold. A deep cleft between the bundles is sufficiently large to accommodate macromolecules such as alginate polysaccharide. This is the first structural report on a bacterial cell-surface alginate-binding protein with an M75 peptidase motif. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Possible implication of bacterial infection in acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation

    Directory of Open Access Journals (Sweden)

    Shigeo eFuji

    2014-04-01

    Full Text Available Graft-versus-host disease (GVHD is still one of the major causes of morbidity and mortality in allogeneic hematopoietic stem cell transplantation (HSCT. In the pathogenesis of acute GVHD, it has been established that donor-derived T cells activated in the recipient play a major role in GVHD in initiation and maintenance within an inflammatory cascade. To reduce the risk of GVHD, intensification of GVHD prophylaxis like T cell depletion is effective, but it inevitably increases the risk of infectious diseases and abrogates beneficial graft-versus-leukemia effects. Although various cytokines are considered to play an important role in the pathogenesis of GVHD, GVHD initiation is such a complex process that cannot be prevented by means of single inflammatory cytokine inhibition. Thus, efficient methods to control the whole inflammatory milieu both on cellular and humoral view are needed. In this context, infectious diseases can theoretically contribute to an elevation of inflammatory cytokines after allogeneic HSCT and activation of various subtypes of immune effector cells, which might in summary lead to an aggravation of acute GVHD. The appropriate treatments or prophylaxis of bacterial infection during the early phase after allogeneic HSCT might be beneficial to reduce not only infectious-related but also GVHD-related mortality. Here, we aim to review the literature addressing the interactions of bacterial infections and GVHD after allogeneic HSCT.

  13. Simulated bacterial infection disrupts the circadian fluctuation of immune cells in wrinkle-lipped bats (Chaerephon plicatus

    Directory of Open Access Journals (Sweden)

    Philipp Weise

    2017-08-01

    Full Text Available Background Leukocyte concentrations follow a circadian pattern in mammals, with elevated values at times of potential contact with pathogens and parasites. We hypothesized that this pattern is disturbed after an immune challenge. Methods In Thailand, we captured wrinkle-lipped bats (Chaerephon plicatus, when they returned to their colony at dawn. We challenged half of the animals (experimental group with bacterial lipopolysaccharides and treated the others only with the carrier liquid (control group. We then compared body mass changes and differences in circulating immune cell counts at 8 h post-treatment. Results In experimental animals, we observed an increase in total leukocyte and neutrophil numbers of 17% and 95%, respectively. In control animals, concentrations of leukocytes decreased by 44% and those of neutrophils remained constant. Experimental treatment had no effect on lymphocytes, yet changes in eosinophil numbers were explained by sex. Eosinophils decreased by 66% in females and by 62% in males. Basophils and monocytes were rarest among all observed cell types and analysis was either impossible because of low numbers or yielded no significant effects, respectively. Discussion Our findings show that a simulated bacterial infection triggered a neutrophil-associated immune response in wrinkle-lipped bats, indicating a disruption of the diurnal fluctuation of immune cells. Our study suggests that bats exhibit circadian rhythms in immune cell counts. The magnitude of these fluctuations may vary across species according to specific-specific infection risks associated with colony sizes or specific roosting habits.

  14. Possible implication of bacterial infection in acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

    Science.gov (United States)

    Fuji, Shigeo; Kapp, Markus; Einsele, Hermann

    2014-01-01

    Graft-versus-host disease (GVHD) is still one of the major causes of morbidity and mortality in allogeneic hematopoietic stem cell transplantation (HSCT). In the pathogenesis of acute GVHD, it has been established that donor-derived T-cells activated in the recipient play a major role in GVHD in initiation and maintenance within an inflammatory cascade. To reduce the risk of GVHD, intensification of GVHD prophylaxis like T-cell depletion is effective, but it inevitably increases the risk of infectious diseases and abrogates beneficial graft-versus-leukemia effects. Although various cytokines are considered to play an important role in the pathogenesis of GVHD, GVHD initiation is such a complex process that cannot be prevented by means of single inflammatory cytokine inhibition. Thus, efficient methods to control the whole inflammatory milieu both on cellular and humoral view are needed. In this context, infectious diseases can theoretically contribute to an elevation of inflammatory cytokines after allogeneic HSCT and activation of various subtypes of immune effector cells, which might in summary lead to an aggravation of acute GVHD. The appropriate treatments or prophylaxis of bacterial infection during the early phase after allogeneic HSCT might be beneficial to reduce not only infectious-related but also GVHD-related mortality. Here, we aim to review the literature addressing the interactions of bacterial infections and GVHD after allogeneic HSCT.

  15. Crystal structure of bacterial cell-surface alginate-binding protein with an M75 peptidase motif

    International Nuclear Information System (INIS)

    Maruyama, Yukie; Ochiai, Akihito; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

    2011-01-01

    Research highlights: → Bacterial alginate-binding Algp7 is similar to component EfeO of Fe 2+ transporter. → We determined the crystal structure of Algp7 with a metal-binding motif. → Algp7 consists of two helical bundles formed through duplication of a single bundle. → A deep cleft involved in alginate binding locates around the metal-binding site. → Algp7 may function as a Fe 2+ -chelated alginate-binding protein. -- Abstract: A gram-negative Sphingomonas sp. A1 directly incorporates alginate polysaccharide into the cytoplasm via the cell-surface pit and ABC transporter. A cell-surface alginate-binding protein, Algp7, functions as a concentrator of the polysaccharide in the pit. Based on the primary structure and genetic organization in the bacterial genome, Algp7 was found to be homologous to an M75 peptidase motif-containing EfeO, a component of a ferrous ion transporter. Despite the presence of an M75 peptidase motif with high similarity, the Algp7 protein purified from recombinant Escherichia coli cells was inert on insulin B chain and N-benzoyl-Phe-Val-Arg-p-nitroanilide, both of which are substrates for a typical M75 peptidase, imelysin, from Pseudomonas aeruginosa. The X-ray crystallographic structure of Algp7 was determined at 2.10 A resolution by single-wavelength anomalous diffraction. Although a metal-binding motif, HxxE, conserved in zinc ion-dependent M75 peptidases is also found in Algp7, the crystal structure of Algp7 contains no metal even at the motif. The protein consists of two structurally similar up-and-down helical bundles as the basic scaffold. A deep cleft between the bundles is sufficiently large to accommodate macromolecules such as alginate polysaccharide. This is the first structural report on a bacterial cell-surface alginate-binding protein with an M75 peptidase motif.

  16. Crystal structure of bacterial cell-surface alginate-binding protein with an M75 peptidase motif

    Energy Technology Data Exchange (ETDEWEB)

    Maruyama, Yukie; Ochiai, Akihito [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Mikami, Bunzo [Laboratory of Applied Structural Biology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Hashimoto, Wataru [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Murata, Kousaku, E-mail: kmurata@kais.kyoto-u.ac.jp [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2011-02-18

    Research highlights: {yields} Bacterial alginate-binding Algp7 is similar to component EfeO of Fe{sup 2+} transporter. {yields} We determined the crystal structure of Algp7 with a metal-binding motif. {yields} Algp7 consists of two helical bundles formed through duplication of a single bundle. {yields} A deep cleft involved in alginate binding locates around the metal-binding site. {yields} Algp7 may function as a Fe{sup 2+}-chelated alginate-binding protein. -- Abstract: A gram-negative Sphingomonas sp. A1 directly incorporates alginate polysaccharide into the cytoplasm via the cell-surface pit and ABC transporter. A cell-surface alginate-binding protein, Algp7, functions as a concentrator of the polysaccharide in the pit. Based on the primary structure and genetic organization in the bacterial genome, Algp7 was found to be homologous to an M75 peptidase motif-containing EfeO, a component of a ferrous ion transporter. Despite the presence of an M75 peptidase motif with high similarity, the Algp7 protein purified from recombinant Escherichia coli cells was inert on insulin B chain and N-benzoyl-Phe-Val-Arg-p-nitroanilide, both of which are substrates for a typical M75 peptidase, imelysin, from Pseudomonas aeruginosa. The X-ray crystallographic structure of Algp7 was determined at 2.10 A resolution by single-wavelength anomalous diffraction. Although a metal-binding motif, HxxE, conserved in zinc ion-dependent M75 peptidases is also found in Algp7, the crystal structure of Algp7 contains no metal even at the motif. The protein consists of two structurally similar up-and-down helical bundles as the basic scaffold. A deep cleft between the bundles is sufficiently large to accommodate macromolecules such as alginate polysaccharide. This is the first structural report on a bacterial cell-surface alginate-binding protein with an M75 peptidase motif.

  17. Biofabrication of morphology improved cadmium sulfide nanoparticles using Shewanella oneidensis bacterial cells and ionic liquid: For toxicity against brain cancer cell lines.

    Science.gov (United States)

    Wang, Li; Chen, Siyuan; Ding, Yiming; Zhu, Qiang; Zhang, Nijia; Yu, Shuqing

    2018-01-01

    The present work determines the anticancer activity of bio-mediated synthesized cadmium sulfide nanoparticles using the ionic liquid and bacterial cells (Shewanella oneidensis). Bacterial cells have been exposed to be important resources that hold huge potential as ecofriendly, cost-effective, evading toxic of dangerous chemicals and the alternative of conventional physiochemical synthesis. The Shewanella oneidensis is an important kind of metal reducing bacterium, known as its special anaerobic respiratory and sulfate reducing capacity. The crystalline nature, phase purity and surface morphology of biosynthesized cadmium sulfide nanoparticles were analyzed by Fourier transform infrared spectroscopy, X-ray diffraction, Field emission scanning electron microscopy, Energy dispersive spectroscopy and Transmission electron microscopy. The use of imidazolium based ionic liquids as soft templating agent for controlling self-assembly and crystal growth direction of metal sulfide nanoparticles has also advanced as an important method. The microscopic techniques showed that the nanoparticles are designed on the nano form and have an excellent spherical morphology, due to the self-assembled mechanism of ionic liquid assistance. The antitumor efficiency of the cadmium sulfide nanoparticles was investigated against brain cancer cell lines using rat glioma cell lines. The effectively improved nano-crystalline and morphological structure of CdS nanoparticles in the presence of IL exhibit excellent cytotoxicity and dispersion ability on the cell shape is completely spread out showing a nice toxic environment against cancer cells. The cytotoxicity effect of cadmium sulfide nanoparticles was discussed with a diagrammatic representation. Copyright © 2017. Published by Elsevier B.V.

  18. An Arabidopsis Homolog of the Bacterial Cell Division Inhibitor SulA Is Involved in Plastid DivisionW⃞

    Science.gov (United States)

    Raynaud, Cécile; Cassier-Chauvat, Corinne; Perennes, Claudette; Bergounioux, Catherine

    2004-01-01

    Plastids have evolved from an endosymbiosis between a cyanobacterial symbiont and a eukaryotic host cell. Their division is mediated both by proteins of the host cell and conserved bacterial division proteins. Here, we identified a new component of the plastid division machinery, Arabidopsis thaliana SulA. Disruption of its cyanobacterial homolog (SSulA) in Synechocystis and overexpression of an AtSulA-green fluorescent protein fusion in Arabidopsis demonstrate that these genes are involved in cell and plastid division, respectively. Overexpression of AtSulA inhibits plastid division in planta but rescues plastid division defects caused by overexpression of AtFtsZ1-1 and AtFtsZ2-1, demonstrating that its role in plastid division may involve an interaction with AtFtsZ1-1 and AtFtsZ2-1. PMID:15208387

  19. Human Neutrophil Clearance of Bacterial Pathogens Triggers Anti-Microbial γδ T Cell Responses in Early Infection

    Science.gov (United States)

    Roberts, Gareth W.; Heuston, Sinéad; Brown, Amanda C.; Chess, James A.; Toleman, Mark A.; Gahan, Cormac G. M.; Hill, Colin; Parish, Tanya; Williams, John D.; Davies, Simon J.; Johnson, David W.; Topley, Nicholas; Moser, Bernhard; Eberl, Matthias

    2011-01-01

    Human blood Vγ9/Vδ2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vγ9/Vδ2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8). In turn, Vγ9/Vδ2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), requires cell-cell contact of Vγ9/Vδ2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1), and results in a TNF-α dependent proliferation of Vγ9/Vδ2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting γδ T cell responses in vitro. Patients with acute peritoneal-dialysis (PD)-associated bacterial peritonitis – characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity – show a selective activation of local Vγ9/Vδ2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The γδ T cell-driven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of γδ T cells and TNF-α and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive γδ T cells in early

  20. Effect of Structure on the Interactions between Five Natural Antimicrobial Compounds and Phospholipids of Bacterial Cell Membrane on Model Monolayers

    Directory of Open Access Journals (Sweden)

    Stella W. Nowotarska

    2014-06-01

    Full Text Available Monolayers composed of bacterial phospholipids were used as model membranes to study interactions of the naturally occurring phenolic compounds 2,5-dihydroxybenzaldehyde and 2-hydroxy-5-methoxybenzaldehyde, and the plant essential oil compounds carvacrol, cinnamaldehyde, and geraniol, previously found to be active against both Gram-positive and Gram-negative pathogenic microorganisms. The lipid monolayers consist of 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE, 1,2-dihexa- decanoyl-sn-glycero-3-phospho-(1'-rac-glycerol (DPPG, and 1,1',2,2'-tetratetradecanoyl cardiolipin (cardiolipin. Surface pressure–area (π-A and surface potential–area (Δψ-A isotherms were measured to monitor changes in the thermodynamic and physical properties of the lipid monolayers. Results of the study indicated that the five compounds modified the three lipid monolayer structures by integrating into the monolayer, forming aggregates of antimicrobial –lipid complexes, reducing the packing effectiveness of the lipids, increasing the membrane fluidity, and altering the total dipole moment in the monolayer membrane model. The interactions of the five antimicrobial compounds with bacterial phospholipids depended on both the structure of the antimicrobials and the composition of the monolayers. The observed experimental results provide insight into the mechanism of the molecular interactions between naturally-occurring antimicrobial compounds and phospholipids of the bacterial cell membrane that govern activities.

  1. Spatial distribution of bacterial communities on volumetric and planar anodes in single-chamber air-cathode microbial fuel cells

    KAUST Repository

    Vargas, Ignacio T.

    2013-05-29

    Pyrosequencing was used to characterize bacterial communities in air-cathode microbial fuel cells across a volumetric (graphite fiber brush) and a planar (carbon cloth) anode, where different physical and chemical gradients would be expected associated with the distance between anode location and the air cathode. As expected, the stable operational voltage and the coulombic efficiency (CE) were higher for the volumetric anode than the planar anode (0.57V and CE=22% vs. 0.51V and CE=12%). The genus Geobacter was the only known exoelectrogen among the observed dominant groups, comprising 57±4% of recovered sequences for the brush and 27±5% for the carbon-cloth anode. While the bacterial communities differed between the two anode materials, results showed that Geobacter spp. and other dominant bacterial groups were homogenously distributed across both planar and volumetric anodes. This lends support to previous community analysis interpretations based on a single biofilm sampling location in these systems. © 2013 Wiley Periodicals, Inc.

  2. S-layer proteins from Lactobacillus sp. inhibit bacterial infection by blockage of DC-SIGN cell receptor.

    Science.gov (United States)

    Prado Acosta, Mariano; Ruzal, Sandra M; Cordo, Sandra M

    2016-11-01

    Many species of Lactobacillus sp. possess Surface(s) layer proteins in their envelope. Among other important characteristics S-layer from Lactobacillus acidophilus binds to the cellular receptor DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; CD209), which is involved in adhesion and infection of several families of bacteria. In this report we investigate the activity of new S-layer proteins from the Lactobacillus family (Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus helveticus and Lactobacillus kefiri) over the infection of representative microorganisms important to human health. After the treatment of DC-SIGN expressing cells with these proteins, we were able to diminish bacterial infection by up to 79% in both gram negative and mycobacterial models. We discovered that pre-treatment of the bacteria with S-layers from Lactobacillus acidophilus and Lactobacillus brevis reduced bacteria viability but also prevent infection by the pathogenic bacteria. We also proved the importance of the glycosylation of the S-layer from Lactobacillus kefiri in the binding to the receptor and thus inhibition of infection. This novel characteristic of the S-layers proteins may contribute to the already reported pathogen exclusion activity for these Lactobacillus probiotic strains; and might be also considered as a novel enzymatic antimicrobial agents to inhibit bacterial infection and entry to host cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Identification of a Supramolecular Functional Architecture of Streptococcus mutans Adhesin P1 on the Bacterial Cell Surface*

    Science.gov (United States)

    Heim, Kyle P.; Sullan, Ruby May A.; Crowley, Paula J.; El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Tang, Wenxing; Besingi, Richard; Dufrene, Yves F.; Brady, L. Jeannine

    2015-01-01

    P1 (antigen I/II) is a sucrose-independent adhesin of Streptococcus mutans whose functional architecture on the cell surface is not fully understood. S. mutans cells subjected to mechanical extraction were significantly diminished in adherence to immobilized salivary agglutinin but remained immunoreactive and were readily aggregated by fluid-phase salivary agglutinin. Bacterial adherence was restored by incubation of postextracted cells with P1 fragments that contain each of the two known adhesive domains. In contrast to untreated cells, glutaraldehyde-treated bacteria gained reactivity with anti-C-terminal monoclonal antibodies (mAbs), whereas epitopes recognized by mAbs against other portions of the molecule were masked. Surface plasmon resonance experiments demonstrated the ability of apical and C-terminal fragments of P1 to interact. Binding of several different anti-P1 mAbs to unfixed cells triggered release of a C-terminal fragment from the bacterial surface, suggesting a novel mechanism of action of certain adherence-inhibiting antibodies. We also used atomic force microscopy-based single molecule force spectroscopy with tips bearing various mAbs to elucidate the spatial organization and orientation of P1 on living bacteria. The similar rupture lengths detected using mAbs against the head and C-terminal regions, which are widely separated in the tertiary structure, suggest a higher order architecture in which these domains are in close proximity on the cell surface. Taken together, our results suggest a supramolecular organization in which additional P1 polypeptides, including the C-terminal segment originally identified as antigen II, associate with covalently attached P1 to form the functional adhesive layer. PMID:25666624

  4. Bacterial single-cell activities along the nutrient availability gradient in a canyon-shaped reservoir: a seasonal study

    Czech Academy of Sciences Publication Activity Database

    Horňák, Karel; Jezbera, Jan; Šimek, Karel

    2010-01-01

    Roč. 60, č. 3 (2010), s. 215-225 ISSN 0948-3055. [Congress of the International Limnological Society /31./. Kapské Město, 15.08.2010-20.08.2010] R&D Projects: GA ČR(CZ) GA206/08/0015; GA ČR(CZ) GAP504/10/1534 Institutional research plan: CEZ:AV0Z60170517 Keywords : reservoir * bacterial activity * leucine and glucose incorporation * HNA and LNA cells Subject RIV: EE - Microbiology, Virology Impact factor: 2.089, year: 2010

  5. Targeting c-kit receptor in neuroblastomas and colorectal cancers using stem cell factor (SCF)-based recombinant bacterial toxins.

    Science.gov (United States)

    Choudhary, Swati; Pardo, Alessa; Rosinke, Reinhard; Batra, Janendra K; Barth, Stefan; Verma, Rama S

    2016-01-01

    Autocrine activation of c-kit (KIT receptor tyrosine kinase) has been postulated to be a potent oncogenic driver in small cell lung cancer, neuroblastoma (NB), and poorly differentiated colorectal carcinoma (CRC). Although targeted therapy involving tyrosine kinase inhibitors (TKIs) such as imatinib mesylate is highly effective for gastrointestinal stromal tumor carrying V560G c-kit mutation, it does not show much potential for targeting wild-type KIT (WT-KIT). Our study demonstrates the role of stem cell factor (SCF)-based toxin conjugates for targeting WT-KIT-overexpressing malignancies such as NBs and CRCs. We constructed SCF-based recombinant bacterial toxins by genetically fusing mutated form of natural ligand SCF to receptor binding deficient forms of Diphtheria toxin (DT) or Pseudomonas exotoxin A (ETA') and evaluated their efficacy in vitro. Efficient targeting was achieved in all receptor-positive neuroblastoma (IMR-32 and SHSY5Y) and colon cancer cell lines (COLO 320DM, HCT 116, and DLD-1) but not in receptor-negative breast carcinoma cell line (MCF-7) thereby proving specificity. While dose- and time-dependent cytotoxicity was observed in both neuroblastoma cell lines, COLO 320DM and HCT 116 cells, only an anti-proliferative effect was observed in DLD-1 cells. We prove that these novel targeting agents have promising potential as KIT receptor tyrosine kinase targeting system.

  6. Staphylococcus aureus-induced G2/M phase transition delay in host epithelial cells increases bacterial infective efficiency.

    Directory of Open Access Journals (Sweden)

    Ludmila Alekseeva

    Full Text Available Staphylococcus aureus is a highly versatile, opportunistic pathogen and the etiological agent of a wide range of infections in humans and warm-blooded animals. The epithelial surface is its principal site of colonization and infection. In this work, we investigated the cytopathic effect of S. aureus strains from human and animal origins and their ability to affect the host cell cycle in human HeLa and bovine MAC-T epithelial cell lines. S. aureus invasion slowed down cell proliferation and induced a cytopathic effect, resulting in the enlargement of host cells. A dramatic decrease in the number of mitotic cells was observed in the infected cultures. Flow cytometry analysis revealed an S. aureus-induced delay in the G2/M phase transition in synchronous HeLa cells. This delay required the presence of live S. aureus since the addition of the heat-killed bacteria did not alter the cell cycle. The results of Western blot experiments showed that the G2/M transition delay was associated with the accumulation of inactive cyclin-dependent kinase Cdk1, a key inducer of mitosis entry, and with the accumulation of unphosphorylated histone H3, which was correlated with a reduction of the mitotic cell number. Analysis of S. aureus proliferation in asynchronous, G1- and G2-phase-enriched HeLa cells showed that the G2 phase was preferential for bacterial infective efficiency, suggesting that the G2 phase delay may be used by S. aureus for propagation within the host. Taken together, our results divulge the potential of S. aureus in the subversion of key cellular processes such as cell cycle progression, and shed light on the biological significance of S. aureus-induced host cell cycle alteration.

  7. Pectin and Xyloglucan Influence the Attachment of Salmonella enterica and Listeria monocytogenes to Bacterial Cellulose-Derived Plant Cell Wall Models.

    Science.gov (United States)

    Tan, Michelle S F; Rahman, Sadequr; Dykes, Gary A

    2016-01-15

    Minimally processed fresh produce has been implicated as a major source of foodborne microbial pathogens globally. These pathogens must attach to the produce in order to be transmitted. Cut surfaces of produce that expose cell walls are particularly vulnerable. Little is known about the roles that different structural components (cellulose, pectin, and xyloglucan) of plant cell walls play in the attachment of foodborne bacterial pathogens. Using bacterial cellulose-derived plant cell wall models, we showed that the presence of pectin alone or xyloglucan alone affected the attachment of three Salmonella enterica strains (Salmonella enterica subsp. enterica serovar Enteritidis ATCC 13076, Salmonella enterica subsp. enterica serovar Typhimurium ATCC 14028, and Salmonella enterica subsp. indica M4) and Listeria monocytogenes ATCC 7644. In addition, we showed that this effect was modulated in the presence of both polysaccharides. Assays using pairwise combinations of S. Typhimurium ATCC 14028 and L. monocytogenes ATCC 7644 showed that bacterial attachment to all plant cell wall models was dependent on the characteristics of the individual bacterial strains and was not directly proportional to the initial concentration of the bacterial inoculum. This work showed that bacterial attachment was not determined directly by the plant cell wall model or bacterial physicochemical properties. We suggest that attachment of the Salmonella strains may be influenced by the effects of these polysaccharides on physical and structural properties of the plant cell wall model. Our findings improve the understanding of how Salmonella enterica and Listeria monocytogenes attach to plant cell walls, which may facilitate the development of better ways to prevent the attachment of these pathogens to such surfaces. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  8. Upregulation of bacterial-specific Th1 and Th17 responses that are enriched in CXCR5+CD4+ T cells in non-small cell lung cancer.

    Science.gov (United States)

    Ma, Qin-Yun; Huang, Da-Yu; Zhang, Hui-Jun; Wang, Shaohua; Chen, Xiao-Feng

    2017-11-01

    The microbial community in the mucosal surfaces is involved in the development of human cancers, including gastric cancer and colorectal cancer. The respiratory tract in the lung also hosts a distinctive microbial community, but the correlation between this community and lung cancer is largely unknown. Here, we examined the Th1 and Th17 responses toward several bacterial antigens, in CD4 + T cells sourced from the peripheral blood (PB), the lung cancer (LC) tissue, and the gastrointestinal (GI) tract of non-small cell lung cancer (NSCLC) patients. Compared to healthy controls, the NSCLC patients presented significantly higher frequencies of Th1 and Th17 cells reacting to Streptococcus salivarius and S. agalactiae, in the PB, LC, and GI tract. Further investigation showed that the upregulation in anti-bacteria response was likely antigen-specific for two reasons. Firstly, the frequencies of Th1 and Th17 cells reacting to Escherichia coli, a typical GI bacterium, were not upregulated in the PB and the LC of NSCLC patients. Secondly, the S. salivarius and S. agalactiae responses could be partially blocked by Tü39, a MHC class II blocking antibody, suggesting that antigen-specific interaction between CD4 + T cells and antigen-presenting cells was required. We also found that S. salivarius and S. agalactiae could potently activate the monocytes to secrete higher levels of interleukin (IL)-6, IL-12, and tumor necrosis factor, which were Th1- and Th17-skewing cytokines. Interestingly, whereas CXCR5 + CD4 + T cells represented Th1 or Th17 cells. Together, these data demonstrated that NSCLC patients presented a significant upregulation of bacterial-specific Th1 and Th17 responses that were enriched in CXCR5 + CD4 + T cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Human Langerhans cells control Th cells via programmed death-ligand 1 in response to bacterial stimuli and nickel-induced contact allergy.

    Directory of Open Access Journals (Sweden)

    Manuel Hitzler

    Full Text Available Langerhans cells (LCs are suspected to initiate inflammatory immune responses to contact allergens and pathogenic bacteria. In chronic infectious diseases, programmed death ligand (PD-L 1 exhibits both inhibitory and costimulatory functions on T cell-mediated activation and tolerance. Here, we investigated the effects of contact allergens and bacterial stimuli on PD-L1 expression in LCs and the effects of altered PD-L1 expression on cytokine release of subsequently cocultured T cells. Monocyte-derived LCs (MoLCs, LCs, and skin sections of patients suffering from allergic contact dermatitis were challenged with nickel and then analyzed for PD-L1 expression by confocal laser scanning microscopy and flow cytometry. In blocking experiments, we found that the release of Th cell specific cytokines was dependent on both stimulation of LCs and inhibition of PD-L1-PD-1 interactions. Stimulation with peptidoglycan (PGN or lipopolysaccharide (LPS and blockage of PD-L1 with a specific antibody triggered the release of high levels of IL-17, IL-22, TNF-α, and IFN-γ in CD4(+T cells. If nickel was used as a stimulus, blockage of PD-L1 led to high amounts of TNF-α and IL-22. A closer look revealed PD-L1-dependent upregulation of IL-17 secretion in FACS-sorted CCR6(+/CCR4(+ T memory cells. In the presence of anti-PD-L1, PGN induced secretion of IFN-γ and IL-17 in total CCR6(+ cells, while nickel triggered secretion of IFN-γ and IL-17 exclusively in CCR6(+/CCR4(+ cells. Our findings suggest that PD-L1 on LCs plays a crucial role in type IV allergic reactions and in response to bacterial stimuli by controlling the nature of inflammatory Th cell responses.

  10. Human Langerhans cells control Th cells via programmed death-ligand 1 in response to bacterial stimuli and nickel-induced contact allergy.

    Science.gov (United States)

    Hitzler, Manuel; Majdic, Otto; Heine, Guido; Worm, Margitta; Ebert, Grit; Luch, Andreas; Peiser, Matthias

    2012-01-01

    Langerhans cells (LCs) are suspected to initiate inflammatory immune responses to contact allergens and pathogenic bacteria. In chronic infectious diseases, programmed death ligand (PD-L) 1 exhibits both inhibitory and costimulatory functions on T cell-mediated activation and tolerance. Here, we investigated the effects of contact allergens and bacterial stimuli on PD-L1 expression in LCs and the effects of altered PD-L1 expression on cytokine release of subsequently cocultured T cells. Monocyte-derived LCs (MoLCs), LCs, and skin sections of patients suffering from allergic contact dermatitis were challenged with nickel and then analyzed for PD-L1 expression by confocal laser scanning microscopy and flow cytometry. In blocking experiments, we found that the release of Th cell specific cytokines was dependent on both stimulation of LCs and inhibition of PD-L1-PD-1 interactions. Stimulation with peptidoglycan (PGN) or lipopolysaccharide (LPS) and blockage of PD-L1 with a specific antibody triggered the release of high levels of IL-17, IL-22, TNF-α, and IFN-γ in CD4(+)T cells. If nickel was used as a stimulus, blockage of PD-L1 led to high amounts of TNF-α and IL-22. A closer look revealed PD-L1-dependent upregulation of IL-17 secretion in FACS-sorted CCR6(+)/CCR4(+) T memory cells. In the presence of anti-PD-L1, PGN induced secretion of IFN-γ and IL-17 in total CCR6(+) cells, while nickel triggered secretion of IFN-γ and IL-17 exclusively in CCR6(+)/CCR4(+) cells. Our findings suggest that PD-L1 on LCs plays a crucial role in type IV allergic reactions and in response to bacterial stimuli by controlling the nature of inflammatory Th cell responses.

  11. Bacterial Keratitis

    Science.gov (United States)

    ... Español Eye Health / Eye Health A-Z Bacterial Keratitis Sections What Is Bacterial Keratitis? Bacterial Keratitis Symptoms ... Lens Care Bacterial Keratitis Treatment What Is Bacterial Keratitis? Leer en Español: ¿Qué Es la Queratitis Bacteriana? ...

  12. Expression of sperm-specific protamines impairs bacterial and eukaryotic cell proliferation.

    Science.gov (United States)

    Günther, Katharina; Paradowska-Dogan, Agnieszka; Bärmann, Birte; Klein, Harald; von Eichel-Streiber, Christoph; Hartley, Ricardo; Weidner, Wolfgang; Behr, Rüdiger; Steger, Klaus

    2015-06-01

    Protamines are the predominant nuclear proteins in testicular spermatids and ejaculated spermatozoa. During spermiogenesis, protamine-DNA interaction induces a higher-order chromatin packaging which finally results in a complete transcriptional stop in elongating spermatids. Although numerous studies investigated the role of protamines in male fertility, to date, no study is available that investigates protamine function, particularly transcriptional silencing, in non-germ cells. Transcriptional stop due to the high binding affinity of arginine-rich protamines to the negatively charged DNA backbone, however, may be induced in somatic cells and may result in suppressing cell division in tumor cells. In the present study, we therefore analyzed whether a protamine-mediated chromatin condensation in somatic cancer cell lines can stop gene expression and arrest cancer cell proliferation. In contrast to terminally differentiated sperm, cancer cells represent immortalized cells that have modulated natural mechanisms for the regulation of apoptosis and cell proliferation. We expressed human protamines in two fast-growing cell systems, E. coli and HeLa cells. In both cases, protamine expression significantly attenuated cell proliferation when compared with control cells. To our knowledge, this is the first study that demonstrates a stop of cell proliferation in both E. coli and HeLa cells by protamine expression. Follow-up studies on the molecular effect of protamines on proliferative cells may, in the future, open new avenues to investigate effective and specific treatments of cancer cells.

  13. Effect of immediate initiation of antiretroviral therapy on risk of severe bacterial infections in HIV-positive people with CD4 cell counts of more than 500 cells per μL

    DEFF Research Database (Denmark)

    O'Connor, Jemma L; Vjecha, Michael J; Phillips, Andrew N

    2017-01-01

    =0·52). These results were consistent when subgroups of the severe bacterial infection composite were analysed separately. INTERPRETATION: Immediate ART reduces the risk of several severe bacterial infections in HIV-positive people with high CD4 cell count. This is partly explained by ART...

  14. Response of single bacterial cells to stress gives rise to complex history dependence at the population level

    Science.gov (United States)

    Mathis, Roland; Ackermann, Martin

    2016-01-01

    Most bacteria live in ever-changing environments where periods of stress are common. One fundamental question is whether individual bacterial cells have an increased tolerance to stress if they recently have been exposed to lower levels of the same stressor. To address this question, we worked with the bacterium Caulobacter crescentus and asked whether exposure to a moderate concentration of sodium chloride would affect survival during later exposure to a higher concentration. We found that the effects measured at the population level depended in a surprising and complex way on the time interval between the two exposure events: The effect of the first exposure on survival of the second exposure was positive for some time intervals but negative for others. We hypothesized that the complex pattern of history dependence at the population level was a consequence of the responses of individual cells to sodium chloride that we observed: (i) exposure to moderate concentrations of sodium chloride caused delays in cell division and led to cell-cycle synchronization, and (ii) whether a bacterium would survive subsequent exposure to higher concentrations was dependent on the cell-cycle state. Using computational modeling, we demonstrated that indeed the combination of these two effects could explain the complex patterns of history dependence observed at the population level. Our insight into how the behavior of single cells scales up to processes at the population level provides a perspective on how organisms operate in dynamic environments with fluctuating stress exposure. PMID:26960998

  15. Use Of Low Light Image Microscopy To Monitor Genetically Engineered Bacterial Luciferase Gene Expression In Living Cells And Gene Activation Throughout The Development Of A Transgenic Organism

    Science.gov (United States)

    Langridge, W. H.; Escher, Alan P.; Baga, M.; O'Kane, Dennis J.; Wampler, John E.; Koncz, C.; Schell, John D.; Szalay, A. A.

    1989-12-01

    Procaryotic and eucaryotic expression vectors which contain a marker gene for selection of transformants linked to genes encoding bacterial luciferase for detection of promoter activated gene expression in vivo were used to transform the appropriate host organisms and drug resistant colonies, cells, or calli were obtained. Bacterial luciferase expression was measured by a luminescence assay for quantitative determination of promoter activation. The cellular localization of bacteria inside the host plant cell cytoplasm was achieved in a single infected plant cell based on the light emitting ability of the genetically engineered bacteria. In addition, the bacterial luciferase marker gene fusions were used to monitor cell type, tissue, and organ specific gene expression in transgenic plants in vivo. To monitor physiological changes during ontogeny of a transformed plant, low light video microscopy, aided by real time image processing techniques developed specifically to enhance extreme low light images, was successfully applied.

  16. Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; Patin, Delphine; Farr, Carol L.; Grant, Joanna C.; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Knuth, Mark W.; Godzik, Adam; Lesley, Scott A.; Elsliger, Marc-André; Deacon, Ashley M.; Wilson, Ian A.

    2015-09-15

    ABSTRACT

    Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.

    IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural

  17. A microtiter plate-based system for the semiautomated growth and assay of bacterial cells for beta-galactosidase activity.

    Science.gov (United States)

    Menzel, R

    1989-08-15

    The introduction of automated pipetting devices, microtiter readers, and microcomputers makes it possible to significantly increase the number of enzyme assays which can be performed as part of the analysis of a biological process. A number of difficulties must be overcome in any such integrated approach based on the microtiter plate. Among these are cell lysis, temperature control, the conversion of microtiter reader optical density values to standard 1-cm path length values, and data management. The utility of such a scheme can be extended to gene regulation and bacterial genetics studies, if bacterial cell culture techniques can be incorporated into the scheme. This paper addresses these issues in the application of a semiautomated system to the study of the induction of the gyrA promoter by treatment (of a gyrA-lac operon fusion-containing strain) with a gyrase inhibitor. This system is specific to the requirements of our studies into the modulation of gene expression by DNA relaxation. The general approach, however, can be readily adapted to other studies.

  18. Effects of inoculation sources on the enrichment and performance of anode bacterial consortia in sensor typed microbial fuel cells

    Directory of Open Access Journals (Sweden)

    Phuong Tran

    2016-01-01

    Full Text Available Microbial fuel cells are a recently emerging technology that promises a number of applications in energy recovery, environmental treatment and monitoring. In this study, we investigated the effect of inoculating sources on the enrichment of electrochemically active bacterial consortia in sensor-typed microbial fuel cells (MFCs. Several MFCs were constructed, operated with modified artificial wastewater and inoculated with different microbial sources from natural soil, natural mud, activated sludge, wastewater and a mixture of those sources. After enrichment, the MFCs inoculated with the natural soil source generated higher and more stable currents (0.53±0.03 mA, in comparisons with the MFCs inoculated with the other sources. The results from denaturing gradient gel electrophoresis (DGGE showed that there were significant changes in bacterial composition from the original inocula to the enriched consortia. Even more interestingly, Pseudomonas sp. was found dominant in the natural soil source and also in the corresponding enriched consortium. The interactions between Pseudomonas sp. and other species in such a community are probably the key for the effective and stable performance of the MFCs.

  19. Lateral flow assay-based bacterial detection using engineered cell wall binding domains of a phage endolysin.

    Science.gov (United States)

    Kong, Minsuk; Shin, Joong Ho; Heu, Sunggi; Park, Je-Kyun; Ryu, Sangryeol

    2017-10-15

    The development of a cost-effective and efficient bacterial detection assay is essential for diagnostic fields, particularly in resource-poor settings. Although antibodies have been widely used for bacterial capture, the production of soluble antibodies is still expensive and time-consuming. Here, we developed a nitrocellulose-based lateral flow assay using cell wall binding domains (CBDs) from phage as a recognition element and colloidal gold nanoparticles as a colorimetric signal for the detection of a model pathogenic bacterium, Bacillus cereus (B. cereus). To improve conjugation efficiency and detection sensitivity, cysteine-glutathione-S-transferase-tagged CBDs and maltose-binding protein-tagged CBDs were produced in Escherichia coli (E. coli) and incorporated in our assays. The sensitivity of the strip to detect B. cereus was 1×10 4 CFU/mL and the overall assay time was 20min. The assay showed superior results compared to the antibody-based approach, and did not show any significant cross-reactivity. This proof of concept study indicates that the lateral flow assay using engineered CBDs hold considerable promise as simple, rapid, and cost-effective biosensors for whole cell detection. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis

    Directory of Open Access Journals (Sweden)

    Jansen Sandra

    2011-02-01

    Full Text Available Abstract Background Recent studies have suggested that the scavenger receptor MARCO (macrophage receptor with collagenous structure mediates activation of the immune response in bacterial infection of the central nervous system (CNS. The chemotactic G-protein-coupled receptor (GPCR formyl-peptide-receptor like-1 (FPRL1 plays an essential role in the inflammatory responses of host defence mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD. Expression of the antimicrobial peptide cathelicidin CRAMP/LL-37 is up-regulated in bacterial meningitis, but the mechanisms underlying CRAMP expression are far from clear. Methods Using a rat meningitis model, we investigated the influence of MARCO and FPRL1 on rCRAMP (rat cathelin-related antimicrobial peptide expression after infection with bacterial supernatants of Streptococcus pneumoniae (SP and Neisseria meningitides (NM. Expression of FPRL1 and MARCO was analyzed by immunofluorescence and real-time RT-PCR in a rat meningitis model. Furthermore, we examined the receptor involvement by real-time RT-PCR, extracellular-signal regulated kinases 1/2 (ERK1/2 phosphorylation and cAMP level measurement in glial cells (astrocytes and microglia and transfected HEK293 cells using receptor deactivation by antagonists. Receptors were inhibited by small interference RNA and the consequences in NM- and SP-induced Camp (rCRAMP gene expression and signal transduction were determined. Results We show an NM-induced increase of MARCO expression by immunofluorescence and real-time RT-PCR in glial and meningeal cells. Receptor deactivation by antagonists and small interfering RNA (siRNA verified the importance of FPRL1 and MARCO for NM- and SP-induced Camp and interleukin-1β expression in glial cells. Furthermore, we demonstrated a functional interaction between FPRL1 and MARCO in NM-induced signalling by real-time RT-PCR, ERK1/2 phosphorylation and cAMP level measurement and show differences between

  1. Surveillance study of bacterial contamination of the parent's cell phone in the NICU and the effectiveness of an anti-microbial gel in reducing transmission to the hands.

    Science.gov (United States)

    Beckstrom, A C; Cleman, P E; Cassis-Ghavami, F L; Kamitsuka, M D

    2013-12-01

    To determine the bacterial contamination rate of the parent's cell phone and the effectiveness of anti-microbial gel in reducing transmission of bacteria from cell phone to hands. Cross-sectional study of cultures from the cell phone and hands before and after applying anti-microbial gel (n=50). All cell phones demonstrated bacterial contamination. Ninety percent had the same bacteria on the cell phone and their cleaned hands. Twenty two percent had no growth on their hands after applying anti-microbial gel after they had the same bacteria on the cell phone and hands. Ninety-two percent of parents were aware that cell phones carried bacteria, but only 38% cleaned their cell phones at least weekly. Bacterial contamination of cell phones may serve as vectors for nosocomial infection in the neonatal intensive care unit. Bacteria transmitted from cell phone to hands may not be eliminated using anti-microbial gel. Development of hand hygiene and cell phone cleaning guidelines are needed regarding bedside cell phone use.

  2. Instant power generation from an air-breathing paper and pencil based bacterial bio-fuel cell.

    Science.gov (United States)

    Veerubhotla, Ramya; Bandopadhyay, Aditya; Das, Debabrata; Chakraborty, Suman

    2015-06-21

    We present a low-cost, disposable microbial fuel cell fabricated on a paper based platform, having a start-up time of 10 s. The platform deploys ordinary pencil strokes for graphite electrode deposition. The device uses a membrane-less design in a one-time injection (OTI) mode or a continuous capillary driven flow mode (CPF), where oxygen from the atmosphere is used up at the cathode for water formation, leading to the generation of bioelectricity. The performance of the fuel cell is evaluated using two bacterial strains, namely, Pseudomonas aeruginosa IIT BT SS1 and Shewanella putrefaciens. This flexible device is shown to retain bacteria for a period of at least one hour, resulting in the generation of almost 0.4 V using P. aeruginosa and a maximum current of 18 μA using S. putrefaciens without the use of any additional catalysts.

  3. Cytokine responses in primary chicken embryo intestinal cells infected with Campylobacter jejuni strains of human and chicken origin and the expression of bacterial virulence-associated genes

    DEFF Research Database (Denmark)

    Li, Yiping; Ingmer, Hanne; Madsen, Mogens

    2008-01-01

    . jejuni strains are capable of invading the CEICs and stimulate these cells in a pro-inflammatory manner and during this interaction the expression of the bacterial virulence-associated genes ciaB, dnaJ and racR is increased. Furthermore, incubation of bacteria with conditioned cell- and bacteria...

  4. Bacterial mitosis

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette

    2003-01-01

    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system...... movement is powered by insertional polymerization of ParM. Consistently, we find that segregating plasmids are positioned at the ends of extending ParM filaments. Thus, the process of R1 plasmid segregation in E. coli appears to be mechanistically analogous to the actin-based motility operating...

  5. Effect of estrogens on bacterial adherence to HeLa cells.

    OpenAIRE

    Sugarman, B; Epps, L R

    1982-01-01

    Incubating confluent cell culture HeLa cells for 18 h with increasing concentrations of estrogens progressively enhanced the subsequent attachment of a variety of radiolabeled bacteria to the HeLa cells. This effect was not caused by other hormones and was not produced by 1-h incubations of HeLa cells or bacteria with hormones. Estrogens did not similarly affect two other receptor cell lines studied. The addition of metabolic inhibitors showed that this effect of estrogens on HeLa cells was e...

  6. Lactobacillus plantarum L9 but not Lactobacillus acidophilus LA reduces tumour necrosis factor induced bacterial translocation in Caco-2 cells.

    Science.gov (United States)

    Wang, B; Chen, J; Wang, S; Zhao, X; Lu, G; Tang, X

    2017-05-30

    Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis and multiple organ dysfunction syndromes. Inflammatory cytokines increase paracellular permeability that allows increased luminal bacteria to translocate across mucosal epithelium and further deteriorate the gut barrier. In order to reduce this risk, the prophylactic use of probiotics has been recently addressed. In this paper, we investigate the protective role toward tumour necrosis factor (TNF)-α induced non-pathogenic Escherichia coli translocation across Caco-2 monolayers of Lactobacillus strains. According to our experimental data, Lactobacillus plantarum L9 and Lactobacillus acidophilus LA have good capacities to adhere to Caco-2 cells. Addition of L. plantarum L9 and L. acidophilus LA to the enterocyte monolayer surface result in significant inhibition of E. coli adhesion and cell internalisation. However, L. plantarum L9 and L. acidophilus LA did not inhibit the growth of the non-pathogenic E. coli B5 after 24 h incubation. Exposure to TNF-α for 6 h caused a dramatic increase in E. coli B5 translocation across Caco-2 cells, which was uncoupled from increases in paracellular permeability. Pretreatment with L. plantarum L9 prevent TNF-α induced transcellular bacterial translocation and IL-8 production in Caco-2 cells. L. plantarum L9 also did not affect the integrity of the monolayers, as indicated by lactate dehydrogenase release, horseradish peroxidase permeability, and transepithelial electrical resistance. L. plantarum L9 showed the potential to protect enterocytes from an acute inflammatory response and therefore could be good potential prophylactic agents in counteracting bacterial translocation.

  7. Performance of Platinum Nanoparticles / Multiwalled Carbon Nanotubes / Bacterial Cellulose Composite as Anode Catalyst for Proton Exchange Membrane Fuel Cells

    Directory of Open Access Journals (Sweden)

    Henry Fonda Aritonang

    2017-05-01

    Full Text Available Highly dispersed platinum (Pt nanoparticles / multiwalled carbon nanotubes (MWCNTs on bacterial cellulose (BC as anode catalysts for proton exchange membrane fuel cells (PEMFC were prepared with various precursors and their electro-catalytic activities towards hydrogen oxidation at 70 oC under non-humidified conditions. The composite was prepared by deposition of Pt nanoparticles and MWCNTs on BC gel by impregnation method using a water solution of metal precursors and MWCNTs followed by reducing reaction using a hydrogen gas. The composite was characterized by using TEM (transmission electron microscopy, EDS (energy dispersive spectroscopy, and XRD (X-ray diffractometry techniques. TEM images and XRD patterns both lead to the observation of spherical metallic Pt nanoparticles with mean diameter of 3-11 nm well impregnated into the BC fibrils. Preliminary tests on a single cell indicate that renewable BC is a good prospect to be explored as a membrane in fuel cell field. Copyright © 2017 BCREC Group. All rights reserved Received: 21st November 2016; Revised: 26th February 2017; Accepted: 27th February 2017 How to Cite: Aritonang, H.F., Kamu, V.S., Ciptati, C., Onggo, D., Radiman, C.L. (2017. Performance of Platinum Nanoparticles / Multiwalled Carbon Nanotubes / Bacterial Cellulose Composite as Anode Catalyst for Proton Exchange Membrane Fuel Cells. Bulletin of Chemical Reaction Engineering & Catalysis, 12 (2: 287-292 (doi:10.9767/bcrec.12.2.803.287-292 Permalink/DOI: http://dx.doi.org/10.9767/bcrec.12.2.803.287-292

  8. Adhesion of bacterial pathogens to soil colloidal particles: influences of cell type, natural organic matter, and solution chemistry.

    Science.gov (United States)

    Zhao, Wenqiang; Walker, Sharon L; Huang, Qiaoyun; Cai, Peng

    2014-04-15

    Bacterial adhesion to granular soil particles is well studied; however, pathogen interactions with naturally occurring colloidal particles (colloids as a function of cell type, natural organic matter (NOM), and solution chemistry. Specifically, batch adhesion experiments were conducted using NOM-present, NOM-stripped soil colloids, Streptococcus suis SC05 and Escherichia coli WH09 over a wide range of solution pH (4.0-9.0) and ionic strength (IS, 1-100 mM KCl). Cell characterization techniques, Freundlich isotherm, and Derjaguin-Landau-Verwey-Overbeek (DLVO) theory (sphere-sphere model) were utilized to quantitatively determine the interactions between cells and colloids. The adhesion coefficients (Kf) of S. suis SC05 to NOM-present and NOM-stripped soil colloids were significantly higher than E. coli WH09, respectively. Similarly, Kf values of S. suis SC05 and E. coli WH09 adhesion to NOM-stripped soil colloids were greater than those colloids with NOM-present, respectively, suggesting NOM inhibits bacterial adhesion. Cell adhesion to soil colloids declined with increasing pH and enhanced with rising IS (1-50 mM). Interaction energy calculations indicate these adhesion trends can be explained by DLVO-type forces, with S. suis SC05 and E. coli WH09 being weakly adhered in shallow secondary energy minima via polymer bridging and charge heterogeneity. S. suis SC05 adhesion decreased at higher IS 100 mM, which is attributed to the change of hydrophobic effect and steric repulsion resulted from the greater presence of extracellular polymeric substances (EPS) on S. suis SC05 surface as compared to E. coli WH09. Hence, pathogen adhesion to the colloidal material is determined by a combination of DLVO, charge heterogeneity, hydrophobic and polymer interactions as a function of solution chemistry. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Denileukin diftitox: a biotherapeutic paradigm shift in the treatment of lymphoid-derived disorders.

    Science.gov (United States)

    Turturro, Francesco

    2007-01-01

    Denileukin diftitox (Ontak) represents an example of a fused molecule that targets cells bearing high affinity interleukin-2 receptors internalized via receptor-mediated endocytosis in an acidified vesicle. Denileukin diftitox is proteolytically cleaved within the endosome liberating the enzymatically active portion of the diphtheria toxin, the A fragment. Diphtheria toxin fragment A is released into the cytosol inhibiting the protein synthesis through the ADP-ribosylation of the elongation factor-2, and leading to cell death. This review focuses on the clinical trials that led to the FDA approval of the drug for cutaneous T cell lymphoma in the US, and investigational studies demonstrating drug-activity against B and T-cell non-Hodgkin's lymphoma, chronic lymphocytic lymphoma and acute graft versus disease within allogeneic hematopoietic stem cell transplant.

  10. Possibilities of immunocytochemical phenotyping of cells in cerebrospinal fluid while children’s bacterial meningitis

    Directory of Open Access Journals (Sweden)

    V. E. Karev

    2013-01-01

    Full Text Available Comparative dynamic clinical and immunomorphological researches of children’s cerebrospinal fluid were made during bacterial purulent meningitis (caused by Neisseria meningitis, Streptococcus pneumoniae, Haemophilus influenza. Regularities connected with presence of CD3 lymphocytes were discovered. They depended on etiology and phase of disease. Children with meningococcal meningitis, pneumococcal meningitis or hemophilic meningitis had different levels of CD3 lymphocytes in cerebrospinal fluid while different phases of disease.

  11. Review of Growth Inhibitory Peptide as a biotherapeutic agent for tumor growth, adhesion, and metastasis.

    Science.gov (United States)

    Muehlemann, M; Miller, K D; Dauphinee, M; Mizejewski, G J

    2005-09-01

    This review surveys the biological activities of an alpha-fetoprotein (AFP) derived peptide termed the Growth Inhibitory Peptide (GIP), which is a synthetic 34 amino acid segment produced from the full length 590 amino acid AFP molecule. The GIP has been shown to be growth-suppressive in both fetal and tumor cells but not in adult terminally-differentiated cells. The mechanism of action of this peptide has not been fully elucidated; however, GIP is highly interactive at the plasma membrane surface in cellular events such as endocytosis, cell contact inhibition and cytoskeleton-induced cell shape changes. The GIP was shown to be growth-suppressive in nine human tumor types and to suppress the spread of tumor infiltrates and metastases in human and mouse mammary cancers. The AFP-derived peptide and its subfragments were also shown to inhibit tumor cell adhesion to extracellular matrix (ECM) proteins and to block platelet aggregation; thus it was expected that the GIP would inhibit cell spreading/migration and metastatic infiltration into host tissues such as lung and pancreas. It was further found that the cyclic versus linear configuration of GIP determined its biological and anti-cancer efficacy. Genbank amino acid sequence identities with a variety of integrin alpha/beta chain proteins supported the GIP's linkage to inhibition of tumor cell adhesion and platelet aggregation. The combined properties of tumor growth suppression, prevention of tumor cell-to-ECM adhesion, and inhibition of platelet aggregation indicate that tumor-to-platelet interactions present promising targets for GIP as an anti-metastatic agent. Finally, based on cholinergic studies, it was proposed that GIP could influence the enzymatic activity of membrane acetylcholinesterases during tumor growth and metastasis. It was concluded that the GIP derived from full-length AFP represents a growth inhibitory motif possessing instrinsic properties that allow it to interfere in cell surface events such

  12. A Novel ZnONPs/PVA-Functionalized Biomaterials for Bacterial Cells Immobilization and its Strengthening Effects on Quinoline Biodegradation.

    Science.gov (United States)

    Jiang, Jinjin; Liu, Yongjun; Liu, Yu; Hou, Siyu

    2018-03-01

    A novel bacterial cells immobilized carrier (ZnONPs/PVA), polyvinyl alcohol (PVA) composites decorated with ZnO nanoparticles (ZnO NPs), was prepared and used for immobilization of the strain Ochrobactrum sp. LC-1, and subsequently for quinoline degrading in water. Characterization of ZnONPs/PVA by using X-ray diffractometer and scanning electron microscopy demonstrated that ZnO NPs were coated on the surface of PVA cubes evenly and the bacterium grew well on the ZnONPs/PVA. Quinoline biodegradation results showed that the degradation effect of quinoline by ZnONPs/PVA immobilized cells was superior to the free cells significantly. The structure and physical properties of ZnNPs/PVA were maintained steady after the reuse of ZnNPs/PVA for cells immobilization several times. Reusability of the ZnONPs/PVA immobilized cells revealed that the quinoline removal ratio was above 97% within 8 h under the conditions of pH neutral, 37 °C when the initial quinoline concentration was 300 mg/L.

  13. Influence of type-I fimbriae and fluid shear stress on bacterial behavior and multicellular architecture of earlyEscherichia colibiofilms at single-cell resolution.

    Science.gov (United States)

    Wang, Liyun; Keatch, Robert; Zhao, Qi; Wright, John A; Bryant, Clare E; Redmann, Anna L; Terentjev, Eugene M

    2018-01-12

    Biofilm formation on abiotic surfaces in food and medical industry can cause severe contamination and infection, yet how biological and physical factors determine cellular architecture of early biofilms and bacterial behavior of the constituent cells remains largely unknown. In this study we examine the specific role of type-I fimbriae in nascent stages of biofilm formation and the response of micro-colonies to environmental flow shear at single-cell resolution. The results show that type-I fimbriae are not required for reversible adhesion from plankton, but critical for irreversible adhesion of Escherichia coli ( E.coli ) MG1655 forming biofilms on polyethylene terephthalate (PET) surfaces. Besides establishing a firm cell-surface contact, the irreversible adhesion seems necessary to initiate the proliferation of E.coli on the surface. After application of shear stress, bacterial retention is dominated by the 3D architecture of colonies independent of the population and the multi-layered structure could protect the embedded cells from being insulted by fluid shear, while cell membrane permeability mainly depends on the biofilm population and the duration time of the shear stress. Importance Bacterial biofilms could lead to severe contamination problems in medical devices and food processing equipment. However, biofilms are usually studied at a rough macroscopic level, thus little is known about how individual bacterial behavior within biofilms and multicellular architecture are influenced by bacterial appendages (e.g. pili/fimbriae) and environmental factors during early biofilm formation. We apply Confocal Laser Scanning Microscopy (CLSM) to visualize E.coli micro-colonies at single-cell resolution. Our findings suggest that type-I fimbriae are vital to the initiation of bacterial proliferation on surfaces and that the responses of biofilm architecture and cell membrane permeability of constituent bacteria to fluid shear stress are different, which are

  14. Differential induction of oxylipin pathway in potato and tobacco cells by bacterial and oomycete elicitors.

    Science.gov (United States)

    Saubeau, Guillaume; Goulitquer, Sophie; Barloy, Dominique; Potin, Philippe; Andrivon, Didier; Val, Florence

    2013-05-01

    Potato and tobacco cells are differentially suited to study oxylipin pathway and elicitor-induced responses. Synthesis of oxylipins via the lipoxygenase (LOX) pathway provides plant cells with an important class of signaling molecules, related to plant stress responses and innate immunity. The aim of this study was to evaluate the induction of LOX pathway in tobacco and potato cells induced by a concentrated culture filtrate (CCF) from Phytophthora infestans and lipopolysaccharide (LPS) from Pectobacterium atrosepticum. Oxylipin activation was evaluated by the measurement of LOX activity and metabolite quantification. The basal levels of oxylipins and fatty acids showed that potato cells contained higher amounts of linoleic (LA), linolenic (LnA) and stearic acids than tobacco cells. The major oxylipin in potato cells, 9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoic acid (9,10,11-THOD), was not detected in tobacco cells. CCF induced a sharp increase of LA and LnA at 8 h in tobacco cells. In contrast they decreased in potato cells. In CCF-treated tobacco cells, colneleic acid increased up to 24 h, colnelenic acid and 9(S)-hydroxyoctadecatrienoic acid (9(S)-HOT) increased up to 16 h. In potato cells, only colneleic acid increased slightly until 16 h. A differential induction of LOX activity was measured in both cells treated by CCF. With LPS treatment, only 9,10,11-THOD accumulation was significantly induced at 16 h in potato cells. Fatty acids were constant in tobacco but decreased in potato cells over the studied time period. These results showed that the two elicitors were differently perceived by the two Solanaceae and that oxylipin pathway is strongly induced in tobacco with the CCF. They also revealed that elicitor-induced responses depended on both cell culture and elicitor.

  15. Advances in analytical methodologies to guide bioprocess engineering for bio-therapeutics.

    Science.gov (United States)

    Saldova, Radka; Kilcoyne, Michelle; Stöckmann, Henning; Millán Martín, Silvia; Lewis, Amanda M; Tuite, Catherine M E; Gerlach, Jared Q; Le Berre, Marie; Borys, Michael C; Li, Zheng Jian; Abu-Absi, Nicholas R; Leister, Kirk; Joshi, Lokesh; Rudd, Pauline M

    2017-03-01

    This study was performed to monitor the glycoform distribution of a recombinant antibody fusion protein expressed in CHO cells over the course of fed-batch bioreactor runs using high-throughput methods to accurately determine the glycosylation status of the cell culture and its product. Three different bioreactors running similar conditions were analysed at the same five time-points using the advanced methods described here. N-glycans from cell and secreted glycoproteins from CHO cells were analysed by HILIC-UPLC and MS, and the total glycosylation (both N- and O-linked glycans) secreted from the CHO cells were analysed by lectin microarrays. Cell glycoproteins contained mostly high mannose type N-linked glycans with some complex glycans; sialic acid was α-(2,3)-linked, galactose β-(1,4)-linked, with core fucose. Glycans attached to secreted glycoproteins were mostly complex with sialic acid α-(2,3)-linked, galactose β-(1,4)-linked, with mostly core fucose. There were no significant differences noted among the bioreactors in either the cell pellets or supernatants using the HILIC-UPLC method and only minor differences at the early time-points of days 1 and 3 by the lectin microarray method. In comparing different time-points, significant decreases in sialylation and branching with time were observed for glycans attached to both cell and secreted glycoproteins. Additionally, there was a significant decrease over time in high mannose type N-glycans from the cell glycoproteins. A combination of the complementary methods HILIC-UPLC and lectin microarrays could provide a powerful and rapid HTP profiling tool capable of yielding qualitative and quantitative data for a defined biopharmaceutical process, which would allow valuable near 'real-time' monitoring of the biopharmaceutical product. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Nitrate reducing bacterial activity in concrete cells of nuclear waste disposal

    Directory of Open Access Journals (Sweden)

    Albrecht A.

    2013-07-01

    Full Text Available Leaching experiments of solid matrices (bitumen and cement pastes have been first implemented to define the physicochemical conditions that microorganisms are likely to meet at the bitumen-concrete interface (see the paper of Bertron et al.. Of course, as might be suspected, the cement matrix imposes highly alkaline pH conditions (10 bacterial strains led us to select Halomonas desiderata as a model bacterium capable of catalyzing the reaction of nitrate reduction in these extreme conditions of pH. The denitrifying activity of Halomonas desiderata was quantified in batch bioreactor in the presence of solid matrices and / or leachate from bitumen and cement matrices. Denitrification was relatively fast in the presence of cement matrix (<100 hours and 2 to 3 times slower in the presence of bituminous matrix. Overall, the presence of solid cement promoted the kinetics of denitrification. The observation of solid surfaces at the end of the experiment revealed the presence of a biofilm of Halomonas desiderata on the cement paste surface. These attached bacteria showed a denitrifying activity comparable to planktonic bacterial culture. On the other side, no colonization of bitumen could be highlighted as either by SEM or epifluorescence microscopy. Now, we are currently developing a continuous experimental bioreactor which should allow us a more rational understanding of the bitumen-cement-microbe interactions.

  17. Nitrate reducing bacterial activity in concrete cells of nuclear waste disposal

    Science.gov (United States)

    Alquier, M.; Kassim, C.; Bertron, A.; Rafrafi, Y.; Sablayrolles, C.; Albrecht, A.; Erable, B.

    2013-07-01

    Leaching experiments of solid matrices (bitumen and cement pastes) have been first implemented to define the physicochemical conditions that microorganisms are likely to meet at the bitumen-concrete interface (see the paper of Bertron et al.). Of course, as might be suspected, the cement matrix imposes highly alkaline pH conditions (10 bacterial strains led us to select Halomonas desiderata as a model bacterium capable of catalyzing the reaction of nitrate reduction in these extreme conditions of pH. The denitrifying activity of Halomonas desiderata was quantified in batch bioreactor in the presence of solid matrices and / or leachate from bitumen and cement matrices. Denitrification was relatively fast in the presence of cement matrix (<100 hours) and 2 to 3 times slower in the presence of bituminous matrix. Overall, the presence of solid cement promoted the kinetics of denitrification. The observation of solid surfaces at the end of the experiment revealed the presence of a biofilm of Halomonas desiderata on the cement paste surface. These attached bacteria showed a denitrifying activity comparable to planktonic bacterial culture. On the other side, no colonization of bitumen could be highlighted as either by SEM or epifluorescence microscopy. Now, we are currently developing a continuous experimental bioreactor which should allow us a more rational understanding of the bitumen-cement-microbe interactions.

  18. Effects of mechanical and bacterial stressors on cytokine and growth-factor expression in periodontal ligament cells.

    Science.gov (United States)

    Proff, P; Reicheneder, C; Faltermeier, A; Kubein-Meesenburg, D; Römer, P

    2014-05-01

    The goal of the study was to examine the effects of a mechanical (orthodontic force simulation by static compressive loading) and a bacterial (endotoxins from a heat-inactivated gram-negative periodontal pathogen) stressor on the expression patterns of factors that are key to regulating osteoclastogenesis and bone remodeling. Three experimental groups were formed with fifth-passage periodontal ligament (PDL) fibroblasts treated by the static application of compressive force (2 g/cm(2)), heat-inactivated aggregatibacter actinomycetemcomitans (1 × 10(7) cells), or both of these stressors combined. Real-time polymerase chain reaction (RT-PCR) was used to study gene expression of IL-6, IL-8, COX-2, IGF-1, VEGF, and MMP-13 in the 3 groups. Protein levels of COX-2, prostaglandin E2 (PGE(2)), and IL-8 production were quantified using immunoblotting and enzyme-linked immunosorbent assay (ELISA). The mechanical stressor upregulated the genes of COX-2, IL-8, IGF-1, and MMP-13 in PDL fibroblasts and the bacterial stressor upregulated IL-6, IL-8, COX-2 and MMP-13. Both stressors in combination upregulated VEGF and caused COX-2 gene expression to increase further; the latter effect was also detected at the protein level and indirectly via the enhanced production of PGE(2). We noted that the posttranscriptional regulation of IL-8 was induced by the mechanical stressor and influenced by PGE(2). While mechanical-stressor application increased the gene expression of COX-2, IL-8, and VEGF in the presence of the bacterial stressor, IL-8 production was posttranscriptionally regulated by the mechanical stressor, whereas COX-2 expression correlated with enhanced production of the inflammatory tissue hormone PGE(2), which exerted a suppressive effect on endotoxin-induced IL-8 production.

  19. Functional genomic studies of uropathogenic Escherichia coli and host urothelial cells when intracellular bacterial communities are assembled.

    Science.gov (United States)

    Reigstad, Christopher S; Hultgren, Scott J; Gordon, Jeffrey I

    2007-07-20

    Uropathogenic Escherichia coli (UPEC), the principal cause of urinary tract infection in women, colonizes the gut as well as the genitourinary tract. Studies of mice inoculated with UTI89, a sequenced isolate, have revealed a complex life cycle that includes formation of intracellular bacterial communities (IBCs) in bladder urothelial cells. To understand how UPEC adapts to life in IBCs, we have used GeneChips and/or quantitative reverse transcriptase PCR to study UTI89 recovered from the distal gut of gnotobiotic mice and from IBCs harvested by laser capture microdissection from the bladder urothelium of infected C3H/HeJ female mice. Host responses were characterized in laser capture microdissected urothelial cells that do or do not contain IBCs. The results reveal components of ferric iron acquisition systems in UTI89 that are expressed at significantly higher levels in IBCs compared with the intestine, including the hemin receptor chuA (1,390 +/- 188-fold). Localized urothelial responses to IBCs help oppose bacterial salvage of host cell iron (e.g. up-regulation of Tfrc (transferrin receptor) and Lcn2 (lipocalin 2)), facilitate glucose import (e.g. Hk2 (hexokinase 2)), and maintain epithelial structural integrity (e.g. Ivl (involucrin) and Sbsn (suprabasin)). DeltachuA mutants produce significantly smaller IBCs compared with wild type UTI89. This difference was not observed in strains lacking sitA (ABC-type iron/manganese transporter subunit), iroN (salmochelin receptor), hlyA (alpha-hemolysin), or entF (enterobactin synthetase subunit). Together, these studies indicate that heme- and siderophore-associated iron play key roles in IBC development and provide a series of microbial and host biomarkers for comparing UPEC strains isolated from humans.

  20. Potencial bioterapêutico dos probióticos nas parasitoses intestinais Probiotics as potential biotherapeutic agents targeting intestinal parasites

    Directory of Open Access Journals (Sweden)

    Teresa Cristina Goulart de Oliveira-Sequeira

    2008-12-01

    Full Text Available Probióticos são microrganismos vivos que, se administrados em quantidades adequadas, promovem benefícios à saúde do homem e dos animais. O crescente interesse nos probióticos fundamenta-se em estudos clínicos nos quais a administração desses organismos foi avaliada na prevenção e no tratamento de desordens intestinais e sistêmicas. Os potenciais mecanismos de ação desses microrganismos incluem a exclusão competitiva, a produção de metabólitos com atividade antimicrobiana e a modulação da resposta imune. Em algumas circunstâncias clínicas específicas, os benefícios produzidos por esses microrganismos foram amplamente documentados, enquanto que em outras os resultados são contraditórios. No presente artigo de revisão, os probióticos foram abordados considerando-se o potencial bioterapêutico desses microrganismos nas parasitoses intestinais.Probiotics are live microorganisms which, when administered in adequate amounts, beneficially affect the general health status of man and animal. The great interest in probiotic microganisms is based on evidences from clinical studies indicating benefits in the prevention or treatment of a broad spectrum of gastrointestinal and systemic disorders. The potential mechanisms by which probiotics beneficially affect health include strengthening of the intestinal barrier, modulation of the immune response, and antagonism of pathogens either by the production of antimicrobial compounds or through competition for mucosal binding sites. In some specific clinical circumstances, there is clear evidence of benefit whereas in others, the results are dubious and important questions remaining unanswered. The aim of this review article is to focus probiotics on their potential as biotherapeutic agents against intestinal parasites.

  1. Architecture and inherent robustness of a bacterial cell-cycle control system.

    Science.gov (United States)

    Shen, Xiling; Collier, Justine; Dill, David; Shapiro, Lucy; Horowitz, Mark; McAdams, Harley H

    2008-08-12

    A closed-loop control system drives progression of the coupled stalked and swarmer cell cycles of the bacterium Caulobacter crescentus in a near-mechanical step-like fashion. The cell-cycle control has a cyclical genetic circuit composed of four regulatory proteins with tight coupling to processive chromosome replication and cell division subsystems. We report a hybrid simulation of the coupled cell-cycle control system, including asymmetric cell division and responses to external starvation signals, that replicates mRNA and protein concentration patterns and is consistent with observed mutant phenotypes. An asynchronous sequential digital circuit model equivalent to the validated simulation model was created. Formal model-checking analysis of the digital circuit showed that the cell-cycle control is robust to intrinsic stochastic variations in reaction rates and nutrient supply, and that it reliably stops and restarts to accommodate nutrient starvation. Model checking also showed that mechanisms involving methylation-state changes in regulatory promoter regions during DNA replication increase the robustness of the cell-cycle control. The hybrid cell-cycle simulation implementation is inherently extensible and provides a promising approach for development of whole-cell behavioral models that can replicate the observed functionality of the cell and its responses to changing environmental conditions.

  2. An X-ray Absorption Fine Structure study of Au adsorbed onto the non-metabolizing cells of two soil bacterial species

    Energy Technology Data Exchange (ETDEWEB)

    Song, Zhen; Kenney, Janice P.L.; Fein, Jeremy B.; Bunker, Bruce A. (Notre)

    2015-02-09

    Gram-positive and Gram-negative bacterial cells can remove Au from Au(III)-chloride solutions, and the extent of removal is strongly pH dependent. In order to determine the removal mechanisms, X-ray Absorption Fine Structure (XAFS) spectroscopy experiments were conducted on non-metabolizing biomass of Bacillus subtilis and Pseudomonas putida with fixed Au(III) concentrations over a range of bacterial concentrations and pH values. X-ray Absorption Near Edge Structure (XANES) and Extended X-ray Absorption Fine Structure (EXAFS) data on both bacterial species indicate that more than 90% of the Au atoms on the bacterial cell walls were reduced to Au(I). In contrast to what has been observed for Au(III) interaction with metabolizing bacterial cells, no Au(0) or Au-Au nearest neighbors were observed in our experimental systems. All of the removed Au was present as adsorbed bacterial surface complexes. For both species, the XAFS data suggest that although Au-chloride-hydroxide aqueous complexes dominate the speciation of Au in solution, Au on the bacterial cell wall is characterized predominantly by binding of Au atoms to sulfhydryl functional groups and amine and/or carboxyl functional groups, and the relative importance of the sulfhydryl groups increases with increasing pH and with decreasing Au loading. The XAFS data for both microorganism species suggest that adsorption is the first step in the formation of Au nanoparticles by bacteria, and the results enhance our ability to account for the behavior of Au in bacteria-bearing geologic systems.

  3. A rapid crosstalk of human gammadelta T cells and monocytes drives the acute inflammation in bacterial infections.

    Directory of Open Access Journals (Sweden)

    Matthias Eberl

    2009-02-01

    Full Text Available Vgamma9/Vdelta2 T cells are a minor subset of T cells in human blood and differ from other T cells by their immediate responsiveness to microbes. We previously demonstrated that the primary target for Vgamma9/Vdelta2 T cells is (E-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP, an essential metabolite produced by a large range of pathogens. Here we wished to study the consequence of this unique responsiveness in microbial infection. The majority of peripheral Vgamma9/Vdelta2 T cells shares migration properties with circulating monocytes, which explains the presence of these two distinct blood cell types in the inflammatory infiltrate at sites of infection and suggests that they synergize in anti-microbial immune responses. Our present findings demonstrate a rapid and HMB-PP-dependent crosstalk between Vgamma9/Vdelta2 T cells and autologous monocytes that results in the immediate production of inflammatory mediators including the cytokines interleukin (IL-6, interferon (IFN-gamma, tumor necrosis factor (TNF-alpha, and oncostatin M (OSM; the chemokines CCL2, CXCL8, and CXCL10; and TNF-related apoptosis-inducing ligand (TRAIL. Moreover, under these co-culture conditions monocytes differentiate within 18 hours into inflammatory dendritic cells (DCs with antigen-presenting functions. Addition of further microbial stimuli (lipopolysaccharide, peptidoglycan induces CCR7 and enables these inflammatory DCs to trigger the generation of CD4(+ effector alphabeta T cells expressing IFN-gamma and/or IL-17. Importantly, our in vitro model replicates the responsiveness to microbes of effluent cells from peritoneal dialysis (PD patients and translates directly to episodes of acute PD-associated bacterial peritonitis, where Vgamma9/Vdelta2 T cell numbers and soluble inflammatory mediators are elevated in patients infected with HMB-PP-producing pathogens. Collectively, these findings suggest a direct link between invading pathogens, microbe

  4. Foxp3(+) T cells regulate immunoglobulin a selection and facilitate diversification of bacterial species responsible for immune homeostasis.

    Science.gov (United States)

    Kawamoto, Shimpei; Maruya, Mikako; Kato, Lucia M; Suda, Wataru; Atarashi, Koji; Doi, Yasuko; Tsutsui, Yumi; Qin, Hongyan; Honda, Kenya; Okada, Takaharu; Hattori, Masahira; Fagarasan, Sidonia

    2014-07-17

    Foxp3(+) T cells play a critical role for the maintenance of immune tolerance. Here we show that in mice, Foxp3(+) T cells contributed to diversification of gut microbiota, particularly of species belonging to Firmicutes. The control of indigenous bacteria by Foxp3(+) T cells involved regulatory functions both outside and inside germinal centers (GCs), consisting of suppression of inflammation and regulation of immunoglobulin A (IgA) selection in Peyer's patches, respectively. Diversified and selected IgAs contributed to maintenance of diversified and balanced microbiota, which in turn facilitated the expansion of Foxp3(+) T cells, induction of GCs, and IgA responses in the gut through a symbiotic regulatory loop. Thus, the adaptive immune system, through cellular and molecular components that are required for immune tolerance and through the diversification as well as selection of antibody repertoire, mediates host-microbial symbiosis by controlling the richness and balance of bacterial communities required for homeostasis. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Microfluidic-Based Droplet and Cell Manipulations Using Artificial Bacterial Flagella

    Directory of Open Access Journals (Sweden)

    Yun Ding

    2016-02-01

    Full Text Available Herein, we assess the functionality of magnetic helical microswimmers as basic tools for the manipulation of soft materials, including microdroplets and single cells. Their ability to perform a range of unit operations is evaluated and the operational challenges associated with their use are established. In addition, we also report on interactions observed between the head of such helical swimmers and the boundaries of droplets and cells and discuss the possibilities of assembling an artificial swimming microorganism or a motorized cell.

  6. Antibacterial compounds of Canadian honeys target bacterial cell wall inducing phenotype changes, growth inhibition and cell lysis that resemble action of β-lactam antibiotics.

    Science.gov (United States)

    Brudzynski, Katrina; Sjaarda, Calvin

    2014-01-01

    Honeys show a desirable broad spectrum activity against Gram-positive and negative bacteria making antibacterial activity an intrinsic property of honey and a desirable source for new drug development. The cellular targets and underlying mechanism of action of honey antibacterial compounds remain largely unknown. To facilitate the target discovery, we employed a method of phenotypic profiling by directly comparing morphological changes in Escherichia coli induced by honeys to that of ampicillin, the cell wall-active β-lactam of known mechanism of action. Firstly, we demonstrated the purity of tested honeys from potential β-lactam contaminations using quantitative LC-ESI-MS. Exposure of log-phase E. coli to honey or ampicillin resulted in time- and concentration-dependent changes in bacterial cell shape with the appearance of filamentous phenotypes at sub-inhibitory concentrations and spheroplasts at the MBC. Cell wall destruction by both agents, clearly visible on microscopic micrographs, was accompanied by increased permeability of the lipopolysaccharide outer membrane as indicated by fluorescence-activated cell sorting (FACS). More than 90% E. coli exposed to honey or ampicillin became permeable to propidium iodide. Consistently with the FACS results, both honey-treated and ampicillin-treated E. coli cells released lipopolysaccharide endotoxins at comparable levels, which were significantly higher than controls (ptransformed with the ampicillin-resistance gene (β-lactamase) remained sensitive to honey, displayed the same level of cytotoxicity, cell shape changes and endotoxin release as ampicillin-sensitive cells. As expected, β-lactamase protected the host cell from antibacterial action of ampicillin. Thus, both honey and ampicillin induced similar structural changes to the cell wall and LPS and that this ability underlies antibacterial activities of both agents. Since the cell wall is critical for cell growth and survival, honey active compounds would be

  7. Atomic model of a cell-wall cross-linking enzyme in complex with an intact bacterial peptidoglycan.

    Science.gov (United States)

    Schanda, Paul; Triboulet, Sébastien; Laguri, Cédric; Bougault, Catherine M; Ayala, Isabel; Callon, Morgane; Arthur, Michel; Simorre, Jean-Pierre

    2014-12-24

    The maintenance of bacterial cell shape and integrity is largely attributed to peptidoglycan, a highly cross-linked biopolymer. The transpeptidases that perform this cross-linking are important targets for antibiotics. Despite this biomedical importance, to date no structure of a protein in complex with an intact bacterial peptidoglycan has been resolved, primarily due to the large size and flexibility of peptidoglycan sacculi. Here we use solid-state NMR spectroscopy to derive for the first time an atomic model of an l,d-transpeptidase from Bacillus subtilis bound to its natural substrate, the intact B. subtilis peptidoglycan. Importantly, the model obtained from protein chemical shift perturbation data shows that both domains-the catalytic domain as well as the proposed peptidoglycan recognition domain-are important for the interaction and reveals a novel binding motif that involves residues outside of the classical enzymatic pocket. Experiments on mutants and truncated protein constructs independently confirm the binding site and the implication of both domains. Through measurements of dipolar-coupling derived order parameters of bond motion we show that protein binding reduces the flexibility of peptidoglycan. This first report of an atomic model of a protein-peptidoglycan complex paves the way for the design of new antibiotic drugs targeting l,d-transpeptidases. The strategy developed here can be extended to the study of a large variety of enzymes involved in peptidoglycan morphogenesis.

  8. Single-Cell Microfluidics to Study the Effects of Genome Deletion on Bacterial Growth Behavior.

    Science.gov (United States)

    Yuan, Xiaofei; Couto, Jillian M; Glidle, Andrew; Song, Yanqing; Sloan, William; Yin, Huabing

    2017-12-15

    By directly monitoring single cell growth in a microfluidic platform, we interrogated genome-deletion effects in Escherichia coli strains. We compared the growth dynamics of a wild type strain with a clean genome strain, and their derived mutants at the single-cell level. A decreased average growth rate and extended average lag time were found for the clean genome strain, compared to those of the wild type strain. Direct correlation between the growth rate and lag time of individual cells showed that the clean genome population was more heterogeneous. Cell culturability (the ratio of growing cells to the sum of growing and nongrowing cells) of the clean genome population was also lower. Interestingly, after the random mutations induced by a glucose starvation treatment, for the clean genome population mutants that had survived the competition of chemostat culture, each parameter markedly improved (i.e., the average growth rate and cell culturability increased, and the lag time and heterogeneity decreased). However, this effect was not seen in the wild type strain; the wild type mutants cultured in a chemostat retained a high diversity of growth phenotypes. These results suggest that quasi-essential genes that were deleted in the clean genome might be required to retain a diversity of growth characteristics at the individual cell level under environmental stress. These observations highlight that single-cell microfluidics can reveal subtle individual cellular responses, enabling in-depth understanding of the population.

  9. Bacterially activated B-cells drive T cell differentiation towards Tr1 through PD-1/PD-L1 expression.

    Science.gov (United States)

    Said, Sawsan Sudqi; Barut, Guliz Tuba; Mansur, Nesteren; Korkmaz, Asli; Sayi-Yazgan, Ayca

    2018-04-01

    Regulatory B cells (Bregs) play a crucial role in immunological tolerance primarily through the production of IL-10 in many diseases including autoimmune disorders, allergy, infectious diseases, and cancer. To date, various Breg subsets with overlapping phenotypes have been identified. However, the roles of Bregs in Helicobacter infection are largely unknown. In the present study, we investigate the phenotype and function of Helicobacter -stimulated B cells. Our results demonstrate that Helicobacter felis -stimulated IL-10- producing B cells (Hf stim - IL-10 + B) are composed of B10 and Transitional 2 Marginal Zone Precursor (T2-MZP) cells with expression of CD9, Tim-1, and programmed death 1 (PD-1). On the other hand, Helicobacter felis -stimulated IL-10- nonproducing B (Hf stim - IL-10 - B) cells are mainly marginal zone (MZ) B cells that express PD-L1 and secrete TGF-β, IL-6, and TNF-α, and IgM and IgG2b. Furthermore, we show that both Hf stim - IL-10 + B cells and Hf stim - IL-10 - B cells induce CD49b + LAG-3 + Tr1 cells. Here, we describe a novel mechanism for PD-1/PD-L1- driven B cell-dependent Tr1 cell differentiation. Finally, we explore the capability of Hf stim - IL-10 - B cells to induce Th17 cell differentiation, which we find to be dependent on TGF-β. Taken together, the current study demonstrates that Hf stim - B cells induce Tr1 cells through the PD-1/PD-L1 axis and Th17 cells by secreting TGF-β. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Modeling of microfluidic microbial fuel cells using quantitative bacterial transport parameters

    Science.gov (United States)

    Mardanpour, Mohammad Mahdi; Yaghmaei, Soheila; Kalantar, Mohammad

    2017-02-01

    The objective of present study is to analyze the dynamic modeling of bioelectrochemical processes and improvement of the performance of previous models using quantitative data of bacterial transport parameters. The main deficiency of previous MFC models concerning spatial distribution of biocatalysts is an assumption of initial distribution of attached/suspended bacteria on electrode or in anolyte bulk which is the foundation for biofilm formation. In order to modify this imperfection, the quantification of chemotactic motility to understand the mechanisms of the suspended microorganisms' distribution in anolyte and/or their attachment to anode surface to extend the biofilm is implemented numerically. The spatial and temporal distributions of the bacteria, as well as the dynamic behavior of the anolyte and biofilm are simulated. The performance of the microfluidic MFC as a chemotaxis assay is assessed by analyzing the bacteria activity, substrate variation, bioelectricity production rate and the influences of external resistance on the biofilm and anolyte's features.

  11. Langerhans cells favor skin flora tolerance through limited presentation of bacterial antigens and induction of regulatory T cells

    NARCIS (Netherlands)

    van der Aar, Angelic M. G.; Picavet, Daisy I.; Muller, Femke J.; de Boer, Leonie; van Capel, Toni M. M.; Zaat, Sebastian A. J.; Bos, Jan D.; Janssen, Hans; George, Thaddeus C.; Kapsenberg, Martien L.; van Ham, S. Marieke; Teunissen, Marcel B. M.; de Jong, Esther C.

    2013-01-01

    The mechanisms preventing detrimental T-cell responses against commensal skin bacteria remain elusive. Using monocyte-derived and skin-derived dendritic cells (DCs), we demonstrate that epidermal Langerhans cells (LCs), the DCs in the most superficial layer of the skin, have a poor capacity to

  12. The genetic and molecular basis of bacterial invasion of epithelial cells

    African Journals Online (AJOL)

    Invasion of epithelial cells was demonstrated to be triggered by invasion plasmid antigens B, C, and D ( IpaB, IpaC and IpaD ) which is accomplished by intracellular spread gene icsA. The invasion of epithelial cells by some individual species of bacteria were also reviewed.Yersinia enterocolitica invasiveness was shown ...

  13. Dispersed cells represent a distinct stage in the transition from bacterial biofilm to planktonic lifestyles

    DEFF Research Database (Denmark)

    Chua, Song Lin; Liu, Yang; Yam, Joey Kuok Hoong

    2014-01-01

    Bacteria assume distinct lifestyles during the planktonic and biofilm modes of growth. Increased levels of the intracellular messenger c-di-GMP determine the transition from planktonic to biofilm growth, while a reduction causes biofilm dispersal. It is generally assumed that cells dispersed from......-dispersing agent, an iron chelator and tobramycin efficiently reduces the survival of the dispersed cells....

  14. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions

    DEFF Research Database (Denmark)

    Schwarz, Sandra; West, T Eoin; Boyer, Frédéric

    2010-01-01

    fluorescens and Serratia proteamaculans-leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly...

  15. Gram-Negative Bacterial Lipopolysaccharide Stimulates Activin A Secretion from Human Amniotic Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Yumiko Abe

    2013-01-01

    Full Text Available Activin A is involved in inflammation. The present study was performed to clarify if lipopolysaccharide, a component of Gram-negative bacteria, stimulates activin A secretion from human amniotic epithelial cells and to determine if activin A plays a role in amnionitis. Fetal membranes were obtained during elective cesarean sections performed in full-term pregnancies of patients without systemic disease, signs of premature delivery, or fetal complications. Amniotic epithelial cells were isolated by trypsinization. The activin A concentrations in the culture media were measured by enzyme-linked immunosorbent assay, and cell proliferation was assessed by 5-bromo-2′-deoxyuridine incorporation. Amniotic epithelial cells secreted activin A in a cell density-dependent manner, and lipopolysaccharide (10 μg/mL enhanced the secretion at each cell density. Lipopolysaccharide (10–50 μg/mL also stimulated activin A secretion in a dose-dependent manner. Contrary to the effect of activin A secretion, lipopolysaccharide inhibited cell proliferation in amniotic epithelial cells. The present study suggests that lipopolysaccharide stimulation of activin A secretion may be a mechanism in the pathogenesis of amnionitis.

  16. A Stochastic Single-Molecule Event Triggers Phenotype Switching of a Bacterial Cell

    Science.gov (United States)

    Xie, Sunney; Choi, Paul; Cai, Long

    2009-03-01

    By monitoring fluorescently labeled lactose permease with single-molecule sensitivity, we investigated the molecular mechanism of how an Escherichia coli cell with the lac operon switches from one phenotype to another. At intermediate inducer concentrations, a population of genetically identical cells exhibits two phenotypes: induced cells with highly fluorescent membranes and uninduced cells with a small number of membrane-bound permeases. We found that this basal-level expression results from partial dissociation of the tetrameric lactose repressor from one of its operators on looped DNA. In contrast, infrequent events of complete dissociation of the repressor from DNA result in large bursts of permease expression that trigger induction of the lac operon. Hence, a stochastic single-molecule event determines a cell's phenotype.

  17. Hyporesponsiveness following booster immunization with bacterial polysaccharides is caused by apoptosis of memory B cells.

    Science.gov (United States)

    Brynjolfsson, Siggeir F; Henneken, Maren; Bjarnarson, Stefania P; Mori, Elena; Del Giudice, Giuseppe; Jonsdottir, Ingileif

    2012-02-01

    Repeated immunizations with polysaccharide (PS) vaccines cause hyporesponsiveness through undefined mechanisms. We assessed the effects of a PS booster on immune responses, frequency, and survival of PS-specific B-cell subpopulations in spleen and bone marrow. Neonatal mice were primed with meningococcus serotype C (MenC) conjugate MenC-CRM(197)+CpG1826, boosted with MenC-CRM(197), MenC-PS, or saline; subsequently, bromodeoxyuridine (BrdU) was injected daily intraperitoneally. MenC-PS-specific cells were labeled with fluorescent MenC-PS and phenotyped by flow cytometry. After MenC-PS booster, proliferating (BrdU(+)) MenC-PS-specific naive B cells (CD138(-)/B220(+); P = .0003) and plasma cells (CD138(+)/B220(-); P = .0002) in spleen were fewer than after saline booster. BrdU(+) MenC-PS-specific plasma cells were also reduced in bone marrow (P = .0308). Compared to saline, MenC-PS booster reduced BrdU(+) IgG(+) MenC-PS-specific B cells in spleen (P = .0002). Twelve hours after the MenC-PS booster, an increased frequency of apoptotic (AnnexinV(+)) MenC-PS-specific B cells in spleen was observed compared with MenC-CRM(197) (P = .0286) or saline (P = .001) boosters. We demonstrated that the MenC-PS booster significantly reduced the frequency of newly activated MenC-PS-specific B cells-mostly switched IgG(+) memory cells-by driving them into apoptosis. It shows directly that apoptosis of PS-specific memory cells is the cause of PS-induced hyporesponsiveness. These results should be taken into account prior to consideration of the use of PS vaccines.

  18. Strategy for Generating Sequence-Defined Aptamer Reagent Sets for Detecting Protein Contaminants in Biotherapeutics.

    Science.gov (United States)

    McGivney, James B; Csordas, Andrew T; Walker, Faye M; Bagley, Elizabeth R; Gruber, Emily M; Mage, Peter L; Casas-Finet, Jose; Nakamoto, Margaret A; Eisenstein, Michael; Larkin, Christopher J; Strouse, Robert J; Soh, H Tom

    2018-03-06

    Biologic drugs are typically manufactured in mammalian host cells, and it is critical from a drug safety and efficacy perspective to detect and remove host cell proteins (HCPs) during production. This is currently achieved with sets of polyclonal antibodies (pAbs), but these suffer from critical shortcomings because their composition is inherently undefined, and they cannot detect nonimmunogenic HCPs. In this work, we report a high-throughput screening and array-based binding characterization strategy that we employed to generate a set of aptamers that overcomes these limitations to achieve sensitive, broad-spectrum detection of HCPs from the widely used Chinese hamster ovary (CHO) cell line. We identified a set of 32 DNA aptamers that achieve better sensitivity than a commercial pAb reagent set and can detect a comparable number of HCPs over a broad range of isoelectric points and sizes. Importantly, these aptamers detect multiple contaminants that are known to be responsible for therapeutic antibody degradation and toxicity in patients. Because HCP aptamer reagents are sequence-defined and chemically synthesized, we believe they may enable safer production of biologic drugs, and this strategy should be broadly applicable for the generation of HCP detection reagents for other cell lines.

  19. Intra-Species Bacterial Quorum Sensing Studied at Single Cell Level in a Double Droplet Trapping System

    Directory of Open Access Journals (Sweden)

    Wilhelm T. S. Huck

    2013-05-01

    Full Text Available In this paper, we investigated the intra-species bacterial quorum sensing at the single cell level using a double droplet trapping system. Escherichia coli transformed to express the quorum sensing receptor protein, LasR, were encapsulated in microdroplets that were positioned adjacent to microdroplets containing the autoinducer, N-(3-oxododecanoyl-L-homoserine lactone (OdDHL. Functional activation of the LasR protein by diffusion of the OdDHL across the droplet interface was measured by monitoring the expression of green fluorescent protein (GFP from a LasR-dependent promoter. A threshold concentration of OdDHL was found to induce production of quorum-sensing associated GFP by E. coli. Additionally, we demonstrated that LasR-dependent activation of GFP expression was also initiated when the adjacent droplets contained single E. coli transformed with the OdDHL synthase gene, LasI, representing a simple quorum sensing circuit between two droplets.

  20. Anti-Inflammatory Effects of Vitamin D on Human Immune Cells in the Context of Bacterial Infection.

    Science.gov (United States)

    Hoe, Edwin; Nathanielsz, Jordan; Toh, Zheng Quan; Spry, Leena; Marimla, Rachel; Balloch, Anne; Mulholland, Kim; Licciardi, Paul V

    2016-12-12

    Vitamin D induces a diverse range of biological effects, including important functions in bone health, calcium homeostasis and, more recently, on immune function. The role of vitamin D during infection is of particular interest given data from epidemiological studies suggesting that vitamin D deficiency is associated with an increased risk of infection. Vitamin D has diverse immunomodulatory functions, although its role during bacterial infection remains unclear. In this study, we examined the effects of 1,25(OH)₂D₃, the active metabolite of vitamin D, on peripheral blood mononuclear cells (PBMCs) and purified immune cell subsets isolated from healthy adults following stimulation with the bacterial ligands heat-killed pneumococcal serotype 19F (HK19F) and lipopolysaccharide (LPS). We found that 1,25(OH)₂D₃ significantly reduced pro-inflammatory cytokines TNF-α, IFN-γ, and IL-1β as well as the chemokine IL-8 for both ligands (three- to 53-fold), while anti-inflammatory IL-10 was increased (two-fold, p = 0.016) in HK19F-stimulated monocytes. Levels of HK19F-specific IFN-γ were significantly higher (11.7-fold, p = 0.038) in vitamin D-insufficient adults (50 nmol/L). Vitamin D also shifted the pro-inflammatory/anti-inflammatory balance towards an anti-inflammatory phenotype and increased the CD14 expression on monocytes ( p = 0.008) in response to LPS but not HK19F stimulation. These results suggest that 1,25(OH)₂D₃ may be an important regulator of the inflammatory response and supports further in vivo and clinical studies to confirm the potential benefits of vitamin D in this context.

  1. Anti-Inflammatory Effects of Vitamin D on Human Immune Cells in the Context of Bacterial Infection

    Directory of Open Access Journals (Sweden)

    Edwin Hoe

    2016-12-01

    Full Text Available Vitamin D induces a diverse range of biological effects, including important functions in bone health, calcium homeostasis and, more recently, on immune function. The role of vitamin D during infection is of particular interest given data from epidemiological studies suggesting that vitamin D deficiency is associated with an increased risk of infection. Vitamin D has diverse immunomodulatory functions, although its role during bacterial infection remains unclear. In this study, we examined the effects of 1,25(OH2D3, the active metabolite of vitamin D, on peripheral blood mononuclear cells (PBMCs and purified immune cell subsets isolated from healthy adults following stimulation with the bacterial ligands heat-killed pneumococcal serotype 19F (HK19F and lipopolysaccharide (LPS. We found that 1,25(OH2D3 significantly reduced pro-inflammatory cytokines TNF-α, IFN-γ, and IL-1β as well as the chemokine IL-8 for both ligands (three- to 53-fold, while anti-inflammatory IL-10 was increased (two-fold, p = 0.016 in HK19F-stimulated monocytes. Levels of HK19F-specific IFN-γ were significantly higher (11.7-fold, p = 0.038 in vitamin D-insufficient adults (<50 nmol/L compared to sufficient adults (>50 nmol/L. Vitamin D also shifted the pro-inflammatory/anti-inflammatory balance towards an anti-inflammatory phenotype and increased the CD14 expression on monocytes (p = 0.008 in response to LPS but not HK19F stimulation. These results suggest that 1,25(OH2D3 may be an important regulator of the inflammatory response and supports further in vivo and clinical studies to confirm the potential benefits of vitamin D in this context.

  2. Shaping bacterial population behavior through computer-interfaced control of individual cells.

    Science.gov (United States)

    Chait, Remy; Ruess, Jakob; Bergmiller, Tobias; Tkačik, Gašper; Guet, Călin C

    2017-11-16

    Bacteria in groups vary individually, and interact with other bacteria and the environment to produce population-level patterns of gene expression. Investigating such behavior in detail requires measuring and controlling populations at the single-cell level alongside precisely specified interactions and environmental characteristics. Here we present an automated, programmable platform that combines image-based gene expression and growth measurements with on-line optogenetic expression control for hundreds of individual Escherichia coli cells over days, in a dynamically adjustable environment. This integrated platform broadly enables experiments that bridge individual and population behaviors. We demonstrate: (i) population structuring by independent closed-loop control of gene expression in many individual cells, (ii) cell-cell variation control during antibiotic perturbation, (iii) hybrid bio-digital circuits in single cells, and freely specifiable digital communication between individual bacteria. These examples showcase the potential for real-time integration of theoretical models with measurement and control of many individual cells to investigate and engineer microbial population behavior.

  3. Investigation of engineered bacterial adhesins for opportunity to interface cells with abiotic materials

    Science.gov (United States)

    Terrell, Jessica L.; Dong, Hong; Holthoff, Ellen L.; Small, Meagan C.; Sarkes, Deborah A.; Hurley, Margaret M.; Stratis-Cullum, Dimitra N.

    2016-05-01

    The convenience of cellular genetic engineering has afforded the power to build `smart' synthetic biological tools with novel applications. Here, we have explored opportunities to hybridize engineered cells with inorganic materials toward the development of 'living' device-compatible systems. Cellular structural biology is engineerable based on the ability to rewrite genetic code to generate recombinant, foreign, or even unnatural proteins. With this capability on the biological end, it should be possible to achieve superior abio-compatibility with the inorganic materials that compose current microfabricated technology. This work investigated the hair-like appendages of Escherichia coli known as Type 1 fimbriae that enable natural adhesion to glycosylated substrates. Sequence alterations within the fimbrial gene cluster were found to be well-tolerated, evidenced by tagging the fimbriae with peptide-based probes. As a further development, fimbriae tips could be reconfigured to, in turn, alter cell binding. In particular, the fimbriae were fused with a genetically optimized peptide-for-inorganics to enable metal binding. This work established methodologies to systematically survey cell adhesion properties across a suite of fimbriae-modified cell types as well as to direct patterned cell adhesion. Cell types were further customized for added complexity including turning on secondary gene expression and binding to gold surfaces. The former demonstrates potential for programmable gene switches and the latter for interfacing biology with inorganic materials. In general, the incorporation of 'programmed' cells into devices can be used to provide the feature of dynamic and automated cell response. The outcomes of this study are foundational toward the critical feature of deliberate positioning of cells as configurable biocomponentry. Overall, cellular integration into bioMEMs will yield advanced sensing and actuation.

  4. Vimentin in Bacterial Infections

    DEFF Research Database (Denmark)

    Mak, Tim N; Brüggemann, Holger

    2016-01-01

    filaments (IFs). IFs have not only roles in maintaining the structural integrity of the cell, but they are also involved in many cellular processes including cell adhesion, immune signaling, and autophagy, processes that are important in the context of bacterial infections. Here, we summarize the knowledge...... about the role of IFs in bacterial infections, focusing on the type III IF protein vimentin. Recent studies have revealed the involvement of vimentin in host cell defenses, acting as ligand for several pattern recognition receptors of the innate immune system. Two main aspects of bacteria......-vimentin interactions are presented in this review: the role of vimentin in pathogen-binding on the cell surface and subsequent bacterial invasion and the interaction of cytosolic vimentin and intracellular pathogens with regards to innate immune signaling. Mechanistic insight is presented involving distinct bacterial...

  5. Dissecting Bacterial Cell Wall Entry and Signaling in Eukaryotic Cells: an Actin-Dependent Pathway Parallels Platelet-Activating Factor Receptor-Mediated Endocytosis

    Directory of Open Access Journals (Sweden)

    Lip Nam Loh

    2017-01-01

    Full Text Available The Gram-positive bacterial cell wall (CW peptidoglycan-teichoic acid complex is released into the host environment during bacterial metabolism or death. It is a highly inflammatory Toll-like receptor 2 (TLR2 ligand, and previous in vivo studies have demonstrated its ability to recapitulate pathological features of pneumonia and meningitis. We report that an actin-dependent pathway is involved in the internalization of the CW by epithelial and endothelial cells, in addition to the previously described platelet-activating factor receptor (PAFr-dependent uptake pathway. Unlike the PAFr-dependent pathway, which is mediated by clathrin and dynamin and does not lead to signaling, the alternative pathway is sensitive to 5-(N-ethyl-N-isopropyl amiloride (EIPA and engenders Rac1, Cdc42, and phosphatidylinositol 3-kinase (PI3K signaling. Upon internalization by this macropinocytosis-like pathway, CW is trafficked to lysosomes. Intracellular CW trafficking is more complex than previously recognized and suggests multiple points of interaction with and without innate immune signaling.

  6. Flow Cytometry Detection of Bacterial Cell Entrapment within the Chitosan Hydrogel and Antibacterial Property of Extracted Chitosan

    Directory of Open Access Journals (Sweden)

    Nafise Sadat Majidi

    2016-09-01

    Full Text Available Background:   Chitosan is unbranched polysaccharide composed of D-glucosamine and N-acetyl-D-glucosamine. Chitosan, derived from shrimp shell, has broad antimicrobial properties against Gram-negative, Gram-positive bacteria and fungi. Methods:  Chitosan was extracted from shrimp shell and studied for cell entrapment and anti-bacterial properties. The hydrogel chitosan was used as the beads for cell entrapment and chitosan beads were designed to deliver cells and nutrients. These data confirmed with flow cytometric analyses.                 Results:   Experimental results exhibited that internal diffusion through the chitosan matrix was the main mechanism for whole gelation by TPP (Tri-polyphosphate. The minimum inhibitory concentration (MIC for chitosan against Staphylococcus aureus and Escherichia coli was 16 and 32 μg/ml respectively. Conclusion:  Despite the antimicrobial properties of chitosan, trapped bacteria in the gel network were alive and were chelated indicating that their access to the outside was limited.

  7. The bacterial tubulin FtsZ requires its intrinsically disordered linker to direct robust cell wall construction.

    Science.gov (United States)

    Sundararajan, Kousik; Miguel, Amanda; Desmarais, Samantha M; Meier, Elizabeth L; Casey Huang, Kerwyn; Goley, Erin D

    2015-06-23

    The bacterial GTPase FtsZ forms a cytokinetic ring at midcell, recruits the division machinery and orchestrates membrane and peptidoglycan cell wall invagination. However, the mechanism for FtsZ regulation of peptidoglycan metabolism is unknown. The FtsZ GTPase domain is separated from its membrane-anchoring C-terminal conserved (CTC) peptide by a disordered C-terminal linker (CTL). Here we investigate CTL function in Caulobacter crescentus. Strikingly, production of FtsZ lacking the CTL (ΔCTL) is lethal: cells become filamentous, form envelope bulges and lyse, resembling treatment with β-lactam antibiotics. This phenotype is produced by FtsZ polymers bearing the CTC and a CTL shorter than 14 residues. Peptidoglycan synthesis still occurs downstream of ΔCTL; however, cells expressing ΔCTL exhibit reduced peptidoglycan crosslinking and longer glycan strands than wild type. Importantly, midcell proteins are still recruited to sites of ΔCTL assembly. We propose that FtsZ regulates peptidoglycan metabolism through a CTL-dependent mechanism that extends beyond simple protein recruitment.

  8. Radiosensitization of hypoxic bacterial cells by nitroimidazoles of low lipophilicity: steady-state and rapid-mix studies

    International Nuclear Information System (INIS)

    Anderson, R.F.; Patel, K.B.; Sehmi, D.S.

    1981-01-01

    Radiosensitization of hypoxic bacterial cells by five 2-nitroimidazoles, with similar reduction potentials to misonidazole but having lower lipophilicites, has been measured in Escherichia coli AB 1157 and Streptococcus lactis 712. Sensitization efficiency progressively decreased with decreasing lepophilicity in E. coli but not in S. lactis. This difference is discussed in terms of the differing membrane properties of the two bacteria; E. coli resembled a multicompartment model, as would also be expected with mammalian cells. Rapid-mix experiments are described which show that the radiosensitization observed after experiments are described which show that the radiosensitization observed after preirradiation contact times between ca. 3 and 30 msec is dependent on the lipophilicity of the sensitizer, higher lipophilicity resulting in a lower contact time being required for radiosensitization. This result and the observation that a highly lipophilic compound affects only half the full oxygen enhancement level after short contact times suggest that part of the sensitization process occurs in a lipophilic compartment of the cell